JP6778200B2 - Compositions Containing Spiranthes sinensis Extract and Pharmaceutical Applications thereof - Google Patents

Description

This application claims the interests of US Provisional Application No. 62 / 106,177 filed on January 21, 2015, and the interests of Taiwan Application No. TW104107161 filed on March 6, 2015. However, these applications are incorporated herein by reference.

Inflammation is the complex biological response of vascular tissue to harmful stimuli such as pathogens, damaged cells, or irritants. The classic signs of acute inflammation are pain, fever, redness, swelling, and loss of function (Lin et al., "Biological evaluation of subglutinol a as a novel immunosuppressive agent for inflammation intervention." ACS Med Chem Lett, Vol. 5 (5). No.), pp. 485-490, 2014). Inflammation is a protective attempt by the organism to eliminate damaging stimuli and to initiate the healing process (Fandino-Vaquero et al., "Orosomucoid secretion levels by epicardial adipose tissue as possible indicator of endothelial dysfunction". in diabetes mellitus or inflammation in coronary artery disease. ”Atherosclerosis Vol. 235 (No. 2): pp. 281-288, 2014). However, it is also worth noting that inflammation can also be harmful to the subject if uncontrolled, for example in an allergic response. In addition, recovery from severe and uncontrolled inflammation usually results in excessive (hyperproliferative) wound healing, which can lead to fibrosis in the tissue and then to cancer development.

Liver fibrosis is a classic example of hyperproliferative wound healing. Liver fibrosis occurs during inflammation of liver tissue and is understood to be involved in cirrhosis and liver cancer. Inflammation is part of the immune system's defense mechanism, but it is important to adequately reduce the degree of inflammation as needed. Liver fibrosis is a scarring process that represents a liver injury response. Liver fibrosis is an excessive (hyperproliferative) wound healing in which excess connective tissue is built in the liver. Extracellular matrix (ECM) is overproduced, poorly degraded, or both. Over time, this process can lead to liver cirrhosis and severe complications, including portal hypertension, liver failure, and liver cancer.
Attempts to treat inflammation and autoimmune disorders have been limited in success. This is partly due to the various inflammation-inducing molecules and numerous inflammation-mediated and inflammatory sensitizing molecules in which the etiology of inflammation and autoimmune disorders appears to elicit inflammation by overlapping mechanisms. Due to the fact that it is a good response. Therefore, compounds, compositions, and methods capable of treating inflammation, neurodegenerative diseases, or autoimmune disorders are highly desirable.

Lin et al., "Biological evaluation of subglutinol a as a novel immunosuppressive agent for inflammation intervention." ACS Med Chem Lett Vol. 5 (No. 5), pp. 485-490, 2014 Fandino-Vaquero et al., "Orosomucoid secretion levels by epicardial adipose tissue as possible indicator of endothelial dysfunction in diabetes mellitus or inflammation in coronary artery disease." Atherosclerosis Vol. 235 (No. 2): pp. 281-288, 2014

Given the above, there is a need for improved compounds to treat inflammation, neurodegenerative diseases, and autoimmune disorders. The present disclosure provides compounds, compositions, and methods that address this need, as well as other advantages.

In one aspect, the disclosure provides a composition formulated for administration to a subject. In some embodiments, the composition comprises cinetylcalol in an amount effective in reducing the inflammatory response. In some embodiments, the composition comprises at least 4.4 μg of cinetylcarol, for example, between 4.4 μg and 44 μg of cinetylcarol. In some embodiments, the composition comprises a Spiranthes sinensis extract. In some embodiments, the reduction of the inflammatory response is induced nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2). ), Interleukin-6 (IL-6), Interleukin-1β (IL-1β), Interleukin 33 (IL-33), NLRP3, Phosphorylated ERK (pERK), NFκB, Matrix metalloproteinase-2 (MMP2) , And reduced expression or activity of one or more biological markers selected from the group consisting of matrix metalloproteinase-9 (MMP9). In some embodiments, the reduction of the inflammatory response is superoxide radical anion (O ₂ ⁻ .), Hydrogen peroxide (H ₂ O ₂ ), hydroxyl radical (.OH), monomorphic oxygen ( ¹ O ₂ ), Inflammatory response, such as one or more free radicals selected from the group consisting of peroxynitrite (ONOO ⁻ ), hydrogen peroxide (NO), nitrosonium cation (NO ⁺ ), and nitroxyl anion (NO ⁻ ). Demonstrated by reduced levels of one or more free radicals in. In some embodiments, the reduction of the inflammatory response is selected from the group consisting of skin, epithelium, synovial tissue, tendons, cartilage, ligaments, bones, muscles, organs, hardeners, blood vessels, bone marrow, and extracellular matrix. Proven by the reduction of tissue swelling, such as in tissue.

In one aspect, the disclosure provides a dosage form containing cinetylcarol formulated for administration to a subject. In some embodiments, the dosage forms are intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous. , Formulated for transmucosal administration, or intraperitoneal administration. In some embodiments, the dosage form is formulated for topical administration. In some embodiments, the dosage form is formulated as a unit dose in liquid, gel, semi-liquid, semi-solid, or solid form. In some embodiments, the dosage form comprises at least 4.4 μg of cinetylcarol, for example between 4.4 μg and 44 μg of cinetylcarol. In some embodiments, the dosage form comprises a Spiranthes sinensis extract. In some embodiments, the dosage form is formulated as a unit dose, such as a unit dose formulated as a food, beverage, or dietary supplement.

In one aspect, the disclosure provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises (a) an amount of Spiranthes sinensis extract (SSE) that is present in an amount effective in reducing the inflammatory response, and (b) a pharmaceutically acceptable carrier. Including. In some embodiments, the reduction in inflammatory response is in the group consisting of iNOS, COX-2, TNF-α, PGE2, IL-6, IL-1β, IL-33, NLRP3, pERK, NFκB, MMP2, and MMP9. Demonstrated by reduced expression or activity of one or more biological markers selected from. In some embodiments, the reduction of the inflammatory response is superoxide radical anion (O ₂ ⁻ .), Hydrogen peroxide (H ₂ O ₂ ), hydroxyl radical (.OH), monomorphic oxygen ( ¹ O ₂ ), Inflammatory response, such as one or more free radicals selected from the group consisting of peroxynitrite (ONOO ⁻ ), hydrogen peroxide (NO), nitrosonium cation (NO ⁺ ), and nitroxyl anion (NO ⁻ ). Demonstrated by reduced levels of one or more free radicals in. In some embodiments, the reduction of the inflammatory response is selected from the group consisting of skin, epithelium, synovial tissue, tendons, cartilage, ligaments, bones, muscles, organs, hardeners, blood vessels, bone marrow, and extracellular matrix. Proven by the reduction of tissue swelling, such as in tissue. In some embodiments, the Spiranthes sinensis extract comprises at least 0.1% by weight cinetylcarol. In some embodiments, 150 g of Spiranthes sinensis extract comprises at least 250 mg of cinetylcarol. In some embodiments, the pharmaceutical composition is formulated for topical administration. In some embodiments, the carrier is intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, Suitable for transmucosal or intraperitoneal administration. In some embodiments, the pharmaceutical composition is formulated as a unit dose in liquid, gel, semi-liquid, semi-solid, or solid form. In some embodiments, the pharmaceutical composition is formulated as a topical cream.

In one aspect, the present disclosure provides a food carrier and a food composition comprising an amount of cinetylcarol, such as at least 44 μg of cinetylcarol. In some compositions, the food composition comprises a Spiranthes sinensis extract. In some embodiments, the food composition is packaged as a beverage, solid food, or semi-solid food. In some embodiments, the food composition is packaged as a food selected from the group consisting of snack bars, cereal products, bakery products, and dairy products.

In one aspect, the disclosure provides a method of inducing antioxidant production. In some embodiments, the method comprises administering to a subject in need thereof a dosage form containing cinetylcarol, the dosage form being formulated for administration to the subject. In some embodiments, the dosage form is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, the dosage form comprises a Spiranthes sinensis extract.

In one aspect, the present disclosure provides a method of reducing the inflammatory response. In some embodiments, the method comprises administering to a subject in need thereof a dosage form containing cinetylcarol, the dosage form being formulated for administration to the subject. In some embodiments, the dosage form is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, the dosage form comprises a Spiranthes sinensis extract.

In one aspect, the present disclosure provides a method of promoting wound healing. In some embodiments, the method comprises administering to a subject in need thereof a dosage form containing cinetylcarol, the dosage form being formulated for administration to the subject. In some embodiments, the dosage form is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, the dosage form comprises a Spiranthes sinensis extract.
Built-in by reference

All publications and patent applications referred to herein are incorporated herein by reference in the same manner as those indicated that individual publications or patent applications are specifically and individually incorporated by reference. ..

1A-B are graphs showing the lack of cytotoxicity of the Spiranthes sinensis extract. RAW264.7 cells were treated with various concentrations of (A) Spiranthes sinensis extract and (B) SI for 24 hours. Cell viability was measured by MTT assay.

2A-C are diagrams showing inhibition of CFA-induced inflammation in mice by treatment with Spiranthes sinensis extract. (A) Change in swelling index of foot (n = 5). The swelling index is calculated by the formula described herein and is expressed as a percentage based on the pre-injection (day 0) swelling index. (B) The area under the curve (AUC) of the 4-day swelling index was calculated and represented in a bar graph. (C) Photograph of mouse foot on day 2. The value is the average of 3 independent experiments ± S. E. It is represented by. ^*** Comparison with P <0.001 CFA monotherapy group. 2A-C are diagrams showing inhibition of CFA-induced inflammation in mice by treatment with Spiranthes sinensis extract. (A) Change in swelling index of foot (n = 5). The swelling index is calculated by the formula described herein and is expressed as a percentage based on the pre-injection (day 0) swelling index. (B) The area under the curve (AUC) of the 4-day swelling index was calculated and represented in a bar graph. (C) Photograph of mouse foot on day 2. The value is the average of 3 independent experiments ± S. E. It is represented by. ^*** Comparison with P <0.001 CFA monotherapy group.

3A-C are diagrams showing inhibition of CFA-induced inflammation in mice with SI treatment. (A) Change in change in swelling index of foot (foot) (n = 5). The swelling index is calculated by the formula described herein and is expressed as a percentage based on the pre-injection (day 0) swelling index. The curve, top to bottom, corresponds to the legend, the group shown from top to bottom, respectively. (B) The area under the curve (AUC) of the swelling index for 4 days was calculated and represented by a bar graph. (C) Photograph of mouse foot on day 2. The value is the average of 3 independent experiments ± S. E. It is represented by. ^** P <0.01, ^*** P <0.001 Comparison with CFA monotherapy group. 3A-C are diagrams showing inhibition of CFA-induced inflammation in mice with SI treatment. (A) Change in change in swelling index of foot (foot) (n = 5). The swelling index is calculated by the formula described herein and is expressed as a percentage based on the pre-injection (day 0) swelling index. The curve, top to bottom, corresponds to the legend, the group shown from top to bottom, respectively. (B) The area under the curve (AUC) of the swelling index for 4 days was calculated and represented by a bar graph. (C) Photograph of mouse foot on day 2. The value is the average of 3 independent experiments ± S. E. It is represented by. ^** P <0.01, ^*** P <0.001 Comparison with CFA monotherapy group.

4A-B are diagrams showing suppression of serum MMP2 and MMP9 after SI treatment in CFA-induced inflamed mice. (A) Gelatin substrate zymography of blood derived from CFA-induced inflamed mice. (B) Quantitative MMP2 and MMP9 from three cohorts of mice. Blood was collected from CFA-induced mice on day 4 and sera were applied to the MMP assay with gelatin substrate zymography. The value is the average of 3 independent experiments ± S. E. Represents. ^** P <0.01, ^*** P <0.001 Comparison with CFA group.

5A-F show that treatment with the Spiranthes sinensis extract reduced cytokine and inflammatory factor mRNA expression in LPS-induced RAW264.7 cells. Various concentrations (5, 20, 50 and 100 μg / mL) of Spiranthes sinensis extracts were used to treat LPS-induced RAW264.7 cells for 6 hours. Amount of mRNA of cytokines (A) IL-1β, (B) IL-6, (C) TNF-α, (D) iNOs, (E) NLRP3 and (F) IL-33 of RAW264.7 cells. mRNA levels were measured by Q-PCR. The value is the average of 3 independent experiments ± S. E. Represents. ^* P <0.05, ^** P <0.01, ^*** P <0.001 Comparison with the LPS treatment-only group of 1 μg / mL.

6A-F show that SI treatment reduced cytokine and inflammatory factor mRNA expression in LPS-induced RAW264.7 cells. Various concentrations (0.1, 1, 5 and 10 μM) of SI were used to treat LPS-induced RAW264.7 cells for 6 hours. Amount of mRNA of cytokines (A) IL-1β, (B) IL-6, (C) TNF-α, (D) iNOS, (E) NLRP3 and (F) IL-33 of RAW264.7 cells. mRNA levels were measured by Q-PCR. The value is the average of 3 independent experiments ± S. E. Represents. ^* P <0.05, ^** P <0.01, ^*** P <0.001 Comparison with the LPS treatment-only group of 1 μg / mL.

7A-D are diagrams illustrating that the Spiranthes sinensis extract inhibits the expression of COX-2, NFκB, p-ERK and ERK in LPS-induced RAW264.7 cells. Treatment of Spiranthes sinensis extracts at various concentrations (5, 20, 50 and 100 μg / mL) was used to treat LPS-induced RAW264.7 cells for 12 hours. Cells were lysed in RIPA buffer for Western blot. Quantification of (A) blot photographs, (B) COX-2, (C) NFκB and (D) p-ERK expression, respectively. The value is the average of 3 independent experiments ± S. E. Represents. ^* P <0.05, ^** P <0.01, ^*** P <0.001 Comparison with LPS group of 1 μg / mL.

8A-D are diagrams showing that SI inhibited the expression of COX-2, NFκB, p-ERK and ERK in RAW264.7 cells. SI treatments of various concentrations (0.1, 1, 5 and 10 μM) were used to treat LPS-induced RAW264.7 cells for 12 hours. Cells were lysed in RIPA buffer for Western blot. Quantification of the expression of (A) blot photographs, (B) COX-2, (C) NFκB, and (D) p-ERK, respectively. The value is the average of 3 independent experiments ± S. E. Represents. ^* P <0.05, ^** P <0.01, ^*** P <0.001 Comparison with LPS group of 1 μg / mL.

9A-B are graphs showing the effects of (A) Spiranthes sinensis extract and (B) SI on NO release of RAW264.7 cells. Various concentrations of Spiranthes sinensis extract (5, 20, 50 and 100 μg / mL) or SI (0.1, 1, 5 and 10 μM) treatments were used to treat LPS-induced RAW264.7 cells for 12 hours. .. Conditional medium was collected and centrifuged to remove cell debris for NO assay. The value is the average of 3 independent experiments ± S. E. Represents. ^** Comparison with LPS group of P <0.01, ^*** P <0.001 1 μg / mL.

FIG. 10 is a graph showing the antioxidant activity of the Spiranthes sinensis extract by the DPPH assay. The value is the average of 3 independent experiments ± S. E. Represents. ^* P <0.05, ^** P <0.01 Comparison with the Spiranthes sinensis extract group of 5 μg / mL.

FIG. 11 is a graph showing cell viability experiments for NHSC and THSC using the MTT assay. Cells were treated with various concentrations of 5, 20, 50 and 100 μg / mL of Spiranthes sinensis extract for 24 hours. The concentration is based on the volume of culture medium.

12A-B are diagrams showing the results of the Oil Red O staining assay under 240X magnification. (A) NHSC, untreated or treated with 5, 20 and 50 μg / mL Spiranthes sinensis extract. (B) THSC, untreated or treated with 5, 20 and 50 μg / mL Spiranthes sinensis extract.

FIG. 13 is a bar graph showing quantification from the results of the experiment shown in FIG.

FIG. 14 is a graph showing the effect of Spiranthes sinensis extract (5, 20, and 50 μg / mL) and curcumin (25 μM) on fatty acid preservation in (A) NHSC and (B) THSC at 48 hours. ^* P <0.05, ^*** p <0.001 Comparison with control group.

15A-B are views showing the results of Sirius red staining of Example 10 under 240X magnification. (A) NHSC, untreated or treated with 5, 20 and 50 μg / mL Spiranthes sinensis extract. (B) THSC, untreated or treated with 5, 20 and 50 μg / mL Spiranthes sinensis extract. The concentration is based on the volume of culture medium.

16A-B show the expression of COL1 I, COL1 III, TGF-βR1 and TGF-βR2 (from left to right in each group of 4 bars) in both (A) NHSC and (B) THSC. It is a graph. 18S is the reference gene in this study. N-CU 25: 25 μM curcumin-treated NHSC, N-CON: untreated NHSC, N-SS 5: 5 μg / mL Spiranthes sinensis extract treated with NHSC, N-SS 20: 20 μg / mL Spiranthes sinensis extract. Treated NHSC, N-SS50: NHSC treated with 50 μg / mL Spiranthes sinensis extract. T-CU 25: 25 μM curcumin treated THSC; T-CON: untreated THSC, T-SS 5: 5 μg / mL Spiranthes sinensis extract treated with THSC, T-SS 20: 20 μg / mL Spiranthes sinensis extract. Treated THSC, T-SS50: THSC treated with 50 μg / mL Spiranthes sinensis extract. The concentration is based on the volume of culture medium. The data represent mean ± SEM from 3 separate experiments.

17A-B are graphs showing the expression of PPARγ in both NHSC and THSC. N-Control: untreated NHSC, N-SS5: NHSC treated with 5 μg / mL Spiranthes sinensis extract, N-SS20: 20 μg / mL NHSC treated with Spiranthes sinensis extract, N-SS 50: 50 μg / mL NHSCs treated with Spiranthes sinensis extract. N-CU25: NHSC treated with 25 μM curcumin. T-Control: untreated THSC, T-SS5: THSC treated with 5 μg / mL Spiranthes sinensis extract, T-SS20: 20 μg / mL THSC treated with Spiranthes sinensis extract, T-SS50: 50 μg / mL THSC treated with a Spiranthes sinensis extract. THSC treated with 25 μM curcumin. Gene expression was standardized with 18S expression. The concentration is based on the volume of culture medium. Data are mean ± S. from three separate experiments. E. Represents.

18A-B are graphs showing the expression of RXRα, NrF2 in both NHSC and THSC. N-Control: untreated NHSC, N-SS5: NHSC treated with 5 μg / mL Spiranthes sinensis extract, N-SS20: 20 μg / mL NHSC treated with Spiranthes sinensis extract, N-SS 50: 50 μg / mL NHSCs treated with Spiranthes sinensis extract. N-CU25: NHSC treated with 25 μM curcumin. T-Control: untreated THSC, T-SS5: THSC treated with 5 μg / mL Spiranthes sinensis extract, T-SS20: 20 μg / mL THSC treated with Spiranthes sinensis extract, T-SS50: 50 μg / mL THSC treated with a Spiranthes sinensis extract. THSC treated with 25 μM curcumin. Gene expression was standardized with 18S expression. The concentration is based on the volume of culture medium. Data are mean ± S. from three separate experiments. E. Represents.

FIG. 19 is an image illustrating wound healing in the dorsal skin of rats in the control group and the Spiranthes sinensis extract (SS) group. Control rats are treated only with sterile gauze. Rats in group SS are treated with 5% Spiranthes sinensis extract in addition to white petrolatum with sterile gauze. Rats in the SS group showed rapid wound healing in response to treatment, and healing was maintained while the wound was healing. Wound healing in SS group rats is faster than wound healing in control rats.

FIG. 20 is a graph showing the wound repair area on the dorsal skin of the control and SS groups during the treatment over a 13-day observation. The repair area (%) is represented by the repair area relative to the wound area.

FIG. 21 is a diagram showing tissue sections of the wound area in the control group and SS group rats. Shown are healing wound sizes in control group (A) and SS treated (B) group animals.

22A-B are diagrams showing tissue sections of the wound area in controls and SS group rats. (A) Healing of skin epithelium and dermis layer in the wound area of control animals. (B) Healing of skin epithelium and dermis layer in the wound area of SS group animals. The image is shown at 16x magnification. 22A-B are diagrams showing tissue sections of the wound area in controls and SS group rats. (A) Healing of skin epithelium and dermis layer in the wound area of control animals. (B) Healing of skin epithelium and dermis layer in the wound area of SS group animals. The image is shown at 16x magnification.

FIG. 23 is a diagram showing a Spiranthes sinensis plant.

FIG. 24 is a graph illustrating the results of the cell viability experiment.

FIG. 25 is a diagram illustrating the results of a real-time PCR assay.

FIG. 26 is a diagram showing a part of a gel showing an inhibitory effect of the Spiranthes sinensis extract on MMP9 activity.

27A-D are diagrams illustrating the results of an assay for MMP2 activity showing an inhibitory effect on the Spiranthes sinensis extract.

Although preferred embodiments of the present disclosure are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely by way of example. One of ordinary skill in the art will immediately come up with numerous modifications, changes and substitutions without departing from this disclosure. It should be understood that various alternatives to the plurality of embodiments of the present disclosure described herein can be used in the practice of the present disclosure. It is intended that the appended claims define the scope of the present disclosure, including methods and structures that fall within the scope of these claims and their equivalents.

Unless otherwise defined, all scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. All patents and publications referred to herein are incorporated by reference.

As used herein and in the claims, the singular forms "one (a)", "one (an)" and "this (the)" are not explicitly defined by context. As long as it includes multiple references.

The terms "effective amount" or "therapeutically effective amount" are sufficient, but not limited to, to provide the intended application, including the treatment of the disease, as defined herein below. Refers to the amount of compound described herein. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the subject and disease condition being treated, such as the weight and age of the subject, the severity of the disease condition, the mode of administration, and the like. , These can be easily determined by those skilled in the art. The term is due to a specific response in the target cell, eg, reduced expression or activity of one or more biological markers such as interleukin-6 (IL-6), NFκB, or phosphorylated ERK (pERK). It is also applied to doses that induce a reduction in the inflammatory response. Specific doses will vary depending on the specific compound selected, the dosage regimen followed, whether administered in combination with other compounds, the timing of administration, the tissue administered, and the physical delivery system of transport. To do.

As used herein, "treating" or "treating" or "palliating" and "ameliorating" are interchangeable herein. Used for. These terms refer to methods for obtaining beneficial or desired results, including but not limited to therapeutic and / or prophylactic benefits. Therapeutic benefit means eradication or improvement of the underlying disorder being treated. Also, therapeutic benefits are achieved by eradicating or improving one or more of the physiological symptoms associated with the underlying disorder, thus improving in the patient even if the patient may still have the underlying disorder. Observed. For prophylactic benefits, the composition may be used in patients at risk of developing a particular disease, or one or more of the physiological symptoms of this disease despite the fact that the disease may not have been diagnosed. Can be administered to patients who have been reported to have.

The term "therapeutic effect" as used herein includes the therapeutic and / or prophylactic benefits described above. Prophylactic effects include delaying or eliminating the onset of the disease or condition, delaying or eliminating the onset of symptoms of the disease or condition, slowing, stopping, or reversing the progression of the disease or condition, or any combination thereof. ..

The terms "co-administration", "administered in combination with", and their grammatical equivalents, as used herein, are two or more. Drugs are administered to animals, thus comprising the simultaneous presence of both drugs and / or their metabolites in the animal. Co-administration includes co-administration as separate compositions, administration at different times as separate compositions, or administration as a composition in which both agents are present.

The term "pharmaceutically acceptable salt" as used herein refers to salts derived from various organic and inorganic pair ions well known in the art, and merely by way of example, sodium, potassium, calcium, etc. Magnesium, ammonium, tetraalkylammonium, etc .; and if the molecule contains basic functionality, salts of organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, mesylate, acetic acid. Examples include salts, maleates and oxalates.

As used herein, "drug" or "biologically active agent" as used herein refers to a biological compound, pharmaceutical compound, or chemical compound or other portion. Non-limiting examples include simple or complex organic or inorganic molecules, peptides, proteins, oligonucleotides, antibodies, antibody derivatives, antibody fragments, vitamin derivatives, carbohydrates, toxins, or chemotherapeutic compounds. Various compounds, such as small molecules and oligomers (eg, oligopeptides and oligonucleotides), and synthetic organic compounds can be synthesized based on different core structures. In addition, various natural sources can result in compounds to be screened, such as plant or animal extracts. One of ordinary skill in the art can easily understand that there are no restrictions on the structural properties of the agents of the present disclosure.

"Signal transduction" is the process by which an stimulating or inhibitory signal is transmitted intracellularly and intracellularly to elicit an intracellular response. A signal transduction pathway modulator refers to a compound that regulates the activity of one or more cellular proteins that are mapped to the same specific signal transduction pathway. Modulators can be those that enhance or suppress the activity of signaling molecules (agonists).

As used herein, the term "inhibition", when referring to a biologically active agent, refers to off-target signaling activity by direct or indirect interaction with the target. Refers to the ability of a drug to reduce compared to activity.

As used herein, "subject" refers to a mammal, such as an animal such as a human. The methods described herein may be useful in both human therapeutics and animal applications. In some embodiments, the patient is a mammal, and in some embodiments, the patient is a human.

As used herein, the term "abnormal immune response" refers to an increased immune response to an antigen, such as an autoigen, and thus inflammation and / or an increased autoimmune or neurodegenerative disease. Refers to an unwanted, excessive, or inappropriate immune response. Abnormal immune responses, as used herein, are characterized by an immune cascade that results in the destruction of body tissues. In general, the consequences of abnormal immunity are not seen in the normal response to infection, but can be triggered by infection. Non-limiting examples of abnormal immune responses include autoimmune disorders, immunodeficiencies and allergies.

As described herein, the terms "inflammation," "inflammatory response," or "immune response" refer to redness, warmth, swelling, pain, and pain that result from increased blood flow and the influx and secretion of immune cells. Means the response of living tissue to damage, infection or irritation, characterized by loss of function.

The term "scar tissue" is formed at the site of damage or disease in any tissue of the body caused by the overproduction of disordered collagen and other connective tissue proteins that act to repair tissue destruction. Means fibrous (fibrous) connective tissue.

The term "regeneration" means the renewal, regrowth, or recovery of a body or body part, tissue, or parenchyma after injury or as a normal body process.

As used herein, a "stem cell" is a differentiated cell in a tissue or organ that is capable of renewing itself and differentiating to give rise to one or more different cell types. Undifferentiated cells found or introduced from external sources such as embryonic stem cells, adult myeloid stem cells.

As used herein, the terms "free radical" and "free radical derivative" include molecules that are considered reactive oxygen species (ROS) and / or reactive nitrogen species (RNS). ROS contains both radical and non-radical species formed as a result of the partial reduction of molecular oxygen (diatomic oxygen; O ₂ ). Non-limiting examples of ROS include superoxide radical anion (O ₂ ⁻ .), Hydrogen peroxide (H ₂ O ₂ ), hydroxyl radical (.OH), single oxygen species ( ¹ O ₂ ), and peroxynite. light (ONOO ^-) and the like. RNS contains both radical and non-radical species formed as a result of the oxidation of L-arginine. Non-limiting examples of RNS include nitric oxide (.NO), nitrosonium cations (NO ⁺ ), nitroxyl anions (NO ⁻ ), and peroxynitrite (ONOO ⁻ ).

As used herein, a biological marker or biomarker generally refers to some measurable indicator of a biological condition or condition. Biological markers are often measured and evaluated to investigate the pharmacological response to normal biological processes, pathogenic processes, or therapeutic interventions.

An "extract" is a substance produced by extracting a portion of a raw material, often by using a solvent such as ethanol or water. The extract may be in the form of a liquid, solid, semi-solid, gel or powder.

As used herein, a "beverage" is a liquid specifically prepared for human ingestion. Beverages generally include several forms of water. Examples of beverages include water, milk, tea, coffee, juices and juice beverages, soft drinks, and sparkling beverages.

A "dietary supplement" is intended to provide nutrients that would otherwise not be ingested in sufficient amounts. Commonly understood supplements include, among other things, vitamins, minerals, fiber, fatty acids, or amino acids. Dietary supplements are defined as foods according to US authorities, but elsewhere, dietary supplements may be classified as drugs or other products.

In various aspects, the present disclosure suffers from disorders associated with abnormal immune responses such as acute and / or chronic inflammatory disorders, liver fibrosis, and autoimmune disorders, including conditions associated with neurodegenerative disorders. With respect to compounds, compositions, and methods for treating an individual. In some embodiments, this is achieved by administering to an individual suffering from the disease a composition comprising a therapeutically effective amount of cinetylcarol or an extract of Spiranthes sinensis. In some embodiments, effective amounts of cinetylcarol and / or extracts of Spiranthes sinensis have anti-inflammatory, anti-liver fibrosis, and antioxidant activity.
Composition

In one aspect, the present disclosure comprises a pharmaceutical composition formulated to administer a composition comprising cinetylcarol (SI) in an effective amount to reduce an inflammatory response to a subject in need thereof. provide. In some embodiments, the pharmaceutical composition comprises cinetylcarol, which has the following chemical structure:

In some embodiments, the disclosed composition comprises a Spiranthes sinensis extract (SSE) derived from the plant Spiranthes sinensis. The Spiranthes sinensis plant is shown in FIG.
Extract

In some embodiments, the subject composition comprises cinetylcarol isolated from an extract of the plant Spiranthes sinensis. In some embodiments, an extract of Spiranthes sinensis is provided. In some embodiments, the extract is an ester extract prepared according to a method comprising the following steps: (a) steps to obtain a plant of Spiranthes sinensis; (b) mixing the plant with a solvent. To obtain the fluid (eg, liquid or supercritical fluid); and (c) the step of drying the fluid to obtain the ester extract of Spiranthes sinensis.

The plant Spiranthes sinensis may be whole plant and includes, but is not limited to, fresh or dried leaves, roots, seeds, bark, fruits, bark, flowers, and / or stems of the plant. , May be any part of the organism Spiranthes sinensis. In some embodiments, the plants of Spiranthes sinensis are dried before proceeding to the extraction step. In some embodiments, the Spiranthes sinensis plant is dried and ground to a suitable size in order to increase the effect of the plant-solvent interaction.

In some embodiments, the Spiranthes sinensis extract is prepared by obtaining a plant of Spiranthes sinensis, mixing the plant with a solvent to obtain a mixture, and then drying the mixture to obtain an extract. .. In some cases, the solvent is ethyl acetate, acetone, and / or n-hexane. In some embodiments, the solvent is ethyl acetate. In some cases, the concentration of ethyl acetate is about 1% -100% (v / v), 5% -100% (v / v), 15% -100% (v / v), 20% -100. % (V / v), 25% -100% (v / v), 30% -100% (v / v), 40% -100% (v / v), 50% -100% (v / v) , 60% -100% (v / v), 70% -100% (v / v), 80% -100% (v / v), 90% -100% (v / v), 10% -90% (V / v), 20% -80% (v / v), 30% -70% (v / v), 40% -60% (v / v), or 25% -75% (v / v) The concentration is based on the total volume of ethyl acetate. In some cases, the solvent is ethyl acetate at a concentration between about 5% and 100% (v / v). In some cases, the concentration of ethyl acetate is between about 10% and 100% (v / v).

In some embodiments, the composition uses an extract of Spiranthes sinensis as a solvent at about 15 ° C to 30 ° C, 20 ° C to 30 ° C, 25 ° C to 30 ° C, 18 ° C to 28 ° C, 20 ° C to 28 ° C, Prepared by mixing at temperatures between 25 ° C. to 28 ° C., 15 ° C. to 37 ° C., 18 ° C. to 37 ° C., 20 ° C. to 37 ° C., or 25 ° C. to 37 ° C. In some cases, mixing is done at 20 ° C to 28 ° C. In some cases, mixing is carried out for approximately 1 day, 2 days, 3 days, 5 days, 6 days, 7 days or more. In some cases, mixing is carried out over approximately 1-2 days.

Preparation may further include drying a mixture of Spiranthes sinensis extract and solvent. Numerous methods are available for drying, for example, drying the mixture can be achieved by vacuum drying, freeze drying, lyophilization, or a combination thereof. In some embodiments, the extract is in the form of the dried powder.

The amount of cinetylcarol and Spiranthes sinensis extract generally correlates with the dry weight of Spiranthes sinensis. For example, the amount of cinetylcarol: Spiranthes sinensis extract: Spiranthes sinensis is about 0.3: 0.1: 1 (w / w / w), about 3: 1:10 (w / w / w), about. It can be in a ratio of 0.5: 0.3: 2 (w / w / w), or about 5: 3: 20 (w / w / w). As a non-limiting example, about 250 mg of cinetylcarol can be separated from about 150 g of the Spiranthes sinensis extract extracted from a dry weight of about 1000 g of Spiranthes sinensis. In some embodiments, the amount of cinetylcarol is at least about 250 mg. In some embodiments, the amount of Spiranthes sinensis extract is at least about 150 g.

In some embodiments, the composition is about 0.0001 to 1000, 0.001 to 100, 0.01 to 100, 0.1 to 100, 1 to 100, based on the total volume of said pharmaceutical composition. , 1.2-100, 1.5-100, 1.8-100, 2-100, 3-100, 4-100, 5-100, 6-100, 7-100, 8-100, 9-100 , 10-100, 30-1000, 50-1000, 80-1000 μg / mL of Spiranthes sinensis extract. In some embodiments, the pharmaceutical composition is about or at least about 0.0001, 0.001, 0.01, 0.1, 1, 1.2, 1 based on the total volume of said pharmaceutical composition. .5, 1.8, 2, 3, 4, 5, 6, 7, 8, 9, 10, 30, 50, 100, 150, 200, 250, 500 μg / mL or more of Spiranthes sinensis extract including.

In some embodiments, the amounts of cinetylcarol or Spiranthes sinensis extract in the compositions of the present disclosure are 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4% 3, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, Less than 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v or v / v.

In some embodiments, the amount of cinetylcarol or Spiranthes sinensis extract is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19. 50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16 .50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75% 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25%, 8%, 7. 75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4 .75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2,75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0 .08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0 .007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0 More than .0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v, or v / v.

In some embodiments, the amounts of cinetylcarol and / or Spiranthes sinensis are 0.0001 to 1000 g, 0.001 to 10 g, 0.005 to 100 g, 0.0005 to 9 g, 0.001 to 8 g, 0. It is in the range of .005-7 g, 0.01-6 g, 0.05-5 g, 0.1-4 g, 0.5-4 g, or 1-3 g. In some embodiments, the amount of cinetylcarol and / or Spiranthes sinensis is at least about 0.0001, 0.001, 0.005, 0.01, 0.05, 0.1, 1, 3, 4 5, 6, 7, 8, 9, 10, 100, 1000 g or more.

The compounds according to the present disclosure are effective over a wide dose range. For example, in the treatment of adult humans, examples of doses that can be used are doses from 0.01 mg to 1000 mg per day, from 0.5 mg to 100 mg, from 1 mg to 50 mg, and from 5 mg to 40 mg per day. Is. An exemplary dose is 10-30 mg per day. The exact dose depends on the route of administration, the mode of administration of the compound, the subject to be treated, the weight of the subject to be treated, and the choice and experience of the attending healthcare professional.

In some embodiments, the disclosure comprises administering to a Spiranthes sinensis extract an effective amount ranging from about 1 to about 100 mg / kg body weight per day. Effective doses are about 0.1 to 80, 0.5 to 80, 1 to 80, 10 to 80, 20 to 80, 50 to 80, 0.1 to 10, 0.5 to 1 kg of body weight per day. It can be in the range of 10, 1-100, 10-100, 20-100, or 50-100 mg. As a non-limiting example, an effective dose can be about 0.01-1.0 mg / kg body weight per day, or 0.06-5 mg / kg body weight per day. As another non-limiting example, an effective dose can be a single dose or a divided dose of about 0.6-5 mg / 60 kg per day. In some cases, the amount of Spiranthes sinensis extract administered to the subject is about 0.6-5 mg per 60 kg body weight per day.
Formulation

In some embodiments, the composition is formulated for administration to a subject in need thereof, and the composition comprises cinetylcarol in an amount effective to reduce the inflammatory response. The composition is formulated in dosage form. In some embodiments, the composition is formulated in a dosage form comprising cinetylcarol. In some embodiments, the composition is formulated in a dosage form comprising an extract of Spiranthes sinensis.

In some embodiments, the composition is about 0.001 to 1000 mg, 0.01 to 100 mg, 0.1 to 200 mg, 3 to 200 mg, 5 to 500 mg, 10 to 100 mg, 10 to 1000 mg, 50 to 200 mg, Alternatively, it contains between 100 and 1000 mg of cinetylcarol. In some embodiments, the pharmaceutical composition is about 0.001 μg or more than about 0.001 μg, about 0.01 μg or more than about 0.01 μg, about 0.1 or more than about 0.1, about 0.5 μg or More than about 0.5 μg, about 1 μg or more than about 1 μg, about 2 μg or more than about 2 μg, about 3 μg or more than about 3 μg, about 4 μg or more than about 4 μg, about 5 μg or more than about 5 μg, about 6 μg or more than about 6 μg, about 7 μg Or more than about 7 μg, about 8 μg or more than about 8 μg, about 9 μg or more than about 9 μg, about 10 μg or more than about 10 μg, about 15 μg or more than about 15 μg, about 20 μg or more than about 20 μg, about 25 μg or more than about 25 μg, about 50 μg or Includes cinetylcarols greater than about 50 μg, greater than about 75 μg or greater than about 75 μg, greater than about 100 μg or greater than about 100 μg, greater than about 200 μg or greater than about 200 μg, greater than about 500 μg or greater than about 500 μg, greater than about 1000 μg or greater than about 1000 μg, or greater. In some embodiments, the pharmaceutical composition comprises at least about 4.4 μg of cinetylcarol. In some embodiments, the pharmaceutical composition comprises an amount between about 4.4 and 44 μg of cinetylcarol.

In some embodiments, the composition provides the compounds of the present disclosure in therapeutically effective amounts as active ingredients, or pharmaceutically acceptable salts, esters, prodrugs, solvates, hydrates or derivatives thereof. It is formulated so that it can be used. If desired, the pharmaceutical composition comprises its pharmaceutically acceptable salts and / or coordination compounds, as well as one or more pharmaceutically acceptable excipients, inert solid diluents and fillers. Contains carriers, sterile aqueous solutions and diluents including various organic solvents, penetration enhancers, solubilizers and adjuvants.

The subject compositions disclosed herein may be formulated for oral administration, intravenous administration, and / or topical administration. In some embodiments, the disclosed pharmaceutical compositions are formulated for topical administration.

In some embodiments, the composition is intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous. Further includes carriers suitable for transmucosal administration, inhalation administration, or intraperitoneal administration.

In some embodiments, the composition is formulated as a unit dose in liquid, gel, semi-liquid, semi-solid, and / or solid form.

In some embodiments, the composition is formulated as a topical cream.

In some embodiments, the composition is formulated as a food product. In some embodiments, the composition is formulated as a beverage. In some embodiments, the composition is formulated as a dietary supplement.
Dosing unit

In one aspect, the disclosure provides a dosage form containing cinetylcarol formulated for administration to a subject. In some embodiments, the dosage form is formulated as a unit dosage form. In some embodiments, the composition is formulated in liquid, gel, semi-liquid, semi-solid, or solid form unit dosage forms. In some embodiments, the unit dosage form may be formulated as a food, beverage, semi-solid diet, semi-liquid diet, and / or dietary supplement. In some embodiments, the dosage form comprises an amount of Spiranthes sinensis extract (SSE) or cinetylcarol that is present in an amount effective in reducing the inflammatory response. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In some embodiments, the dosage form is approximately 0.1 to 1000 μg, 0.1 to 5 μg, 0.1 to 10 μg, 0.1 to 20 μg, 0.1 to 30 μg, 0 of the extracted cinetylcarol. .1 to 40 μg, 0.1 to 50 μg, 0.1 to 100 μg, 0.5 to 100 μg, 1 to 100 μg, 5 to 100 μg, 10 to 100 μg, 20 to 100 μg, or 50 to 100 μg. In some embodiments, the dosage form is at least about 0.1 μg or more than about 0.1 μg, at least about 5 μg or more than about 5 μg, at least about 10 μg or more than about 10 μg, at least about 20 μg or more than about 20 μg, at least about 30 μg. Or it comprises more than about 30 μg, at least about 40 μg or more than about 40 μg, at least about 50 μg or more than about 50 μg, at least about 100 μg or more than about 100 μg, at least about 1000 μg or more than about 1000 μg, or more cinetylcarol. In some embodiments, the dosage of cinetylcarol is between about 4.4 μg and 44 μg. In some embodiments, the amount of cinetylcarol is at least about 4.4 μg. In some embodiments, the unit dose comprises a Spiranthes sinensis extract. In some embodiments, the amount of Spiranthes sinensis extract is at least about 44 μg.

In some embodiments, the unit dosage form is a Spiranthes sinensis extract, based on the total volume of the pharmaceutical composition, from about 0.1 to 1000 μg / mL, 0.1 to 100 μg / mL, 0.5 to. 100 μg / mL, 1-100 μg / mL, 2-100 μg / mL, 5-100 μg / mL, 10-100 μg / mL, 20-100 μg / mL, 50-100 μg / mL, 0.1 μg-150 μg / mL, 0. 5 to 150 μg / mL, 1 to 150 μg / mL, 2 to 150 μg / mL, 5 to 150 μg / mL, 10 to 150 μg / mL, 20 to 150 μg / mL, 50 to 150 μg / mL, 0.1 to 200 μg / mL, Included in amounts between 0.5-200 μg / mL, 1-20 μg / mL, 2-200 μg / mL, 5-200 μg / mL, 10-200 μg / mL, 20-200 μg / mL, or 50-1000 μg / mL .. In some embodiments, the unit dosage of the Spiranthes sinensis extract is at least about 0.1 μg / mL or more than about 0.1 μg / mL, at least about 1 μg / mL or more than about 1 μg / mL, at least about 2 μg / mL. Or more than about 2 μg / mL, at least about 5 μg / mL or more than about 5 μg / mL, at least about 10 μg / mL or more than about 10 μg / mL, at least about 20 μg / mL or more than about 20 μg / mL, at least about 50 μg / mL or about More than 50 μg / mL, at least about 100 μg / mL or about 100 μg / mL, at least about 150 μg / mL or more than about 150 μg / mL, at least about 200 μg / mL or more than about 200 μg / mL, at least about 500 μg / mL or about 500 μg / More than mL, at least about 1000 μg / mL or more than about 1000 μg / mL, or more. In some embodiments, the disclosed composition comprises a Spiranthes sinensis extract between about 5-100 μg / mL.

The amount of compound administered depends on the mammal being treated, the severity of the disorder or condition, the rate of administration, the pharmacokinetics of the compound and the discretion of the prescribing healthcare professional. In some embodiments, the compounds of the present disclosure are administered in a single dose. Single doses of the compounds of the present disclosure can also be used to treat acute conditions. In some embodiments, the compounds of the present disclosure are administered in multiple doses. Dosing may be about once, twice, three times, four times, five times, six times, or more than six times per day. Dosing may be about once a month, once every two weeks, once a week, or once every two days. In another embodiment, the compound of the present disclosure and another agent are administered together from about 1 time per day to about 6 times per day. In another embodiment, administration of the compounds and agents of the present disclosure is continued for less than about 7 days. In yet another embodiment, administration is continued for more than about 6 days, more than 10 days, more than about 14 days, more than about 28 days, more than about 2 months, more than about 6 months, or more than about 1 year. In some cases, achieve and maintain continuous dosing as needed.

Administration of the agents of the present disclosure can be continued as long as necessary. In some embodiments, the agents of the present disclosure are administered over 1 day, over 2 days, over 3 days, over 4 days, over 5 days, over 6 days, over 7 days, over 14 days, over 28 days or more. Administer over. In some embodiments, the agents of the present disclosure are administered for less than 28 days, less than 14 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day. To do. In some embodiments, the agents of the present disclosure are administered continuously over an extended period of time, eg, to treat chronic effects.

Effective amounts of the compounds of the present disclosure, in either single or multiple doses, are intravenous, intraperitoneal, parenteral, by rectal, buccal, intranasal and transdermal, intramuscular injections, It can be administered by any of the acceptable forms of administration of drugs of similar utility, including intramuscular, subcutaneous, oral, topical, or as an inhalant.

If the compound of the present disclosure is administered in a composition comprising one or more agents and the half-life of the agent is shorter than that of the compound of the present disclosure, then the agent and the compound dosage form of the present disclosure will be administered accordingly. Can be adjusted.

With respect to the treatment of autoimmune diseases, the subject compounds or pharmaceutical compositions are, but are not limited to, Enbrel®, Remicade®, Humira®, Avonex®, and Rebif (registered trademarks). It can be used in combination with commonly prescribed drugs, including registered trademarks). For the treatment of respiratory diseases, subject compounds or pharmaceutical compositions include, but are not limited to, Xolair®, Advair®, Singular®, and Spiriva®. , Can be administered in combination with commonly prescribed drugs.

The compounds of the present disclosure may be formulated or administered in combination with other agents that act to reduce the symptoms of inflammatory conditions such as encephalomyelitis, asthma, and other diseases described herein. it can. These agents include non-steroidal anti-inflammatory drugs (NSAIDs), such as acetylsalicylic acid; ibuprofen; naproxen; indomethacin; nabumetone; tolmetin; and the like. Corticosteroids are used to reduce inflammation and suppress the activity of the immune system. The most commonly prescribed drug of this type is prednisone. Chloroquine (Aralen) or hydroxychloroquine (Plaquenil) can also be very useful in some individuals with lupus. These are most often prescribed for lupus skin and joint symptoms. Azathioprine (Imuran) and cyclophosphamide (Cytoxan) tend to suppress inflammation and suppress the immune system. Other drugs, such as methotrexate and cyclosporine, are used to control the symptoms of lupus. Anticoagulants are used to prevent the blood from clotting rapidly. These range from very low doses of aspirin, which prevents platelets from sticking, to heparin / kumadin.

The compounds described herein can be formulated or administered in conjunction with a liquid or solid tissue barrier also known as a lubricant. Examples of tissue barriers include, but are not limited to, polysaccharides, polyglycans, seprafilms, interseeds and hyaluronic acid.

As a medicament that can be administered in combination with the compounds described herein, any suitable drug usefully delivered by inhalation, such as an analgesic, such as codeine, dihydromorphine, ergotamine, fentanyl or morphine; Preparations for angina, eg, zirchiazem; antiallergic agents, eg cromoglycic acid, ketotiphen or nedocromyl; anti-infective agents, eg, cephalosporin, penicillin, streptomycin, sulfonamide, tetracycline or pentamidin; antihistamine agents, eg , Metapyrylene; anti-inflammatory agents such as becromethasone, flunisolide, budesonide, tipredan, triamsinolone acetonide or fluticazone; antitussives such as noscapin; bronchial dilators such as ephedrine, adrenaline, phenotelol, formoterol, isoprenaline, metaproterenol, Fenirefrin, phenylpropanolamine, pyrubuterol, leproterol, limiterol, salbutamol, salmeterol, terbutalin, isoetaline, turobterol, orciprenaline or (-)-4-amino-3,5-dichloro-α-[[[6- [2- (2) -Pyridinyl) ethoxy] hexyl] -amino] methyl] benzenemethanol; diuretics such as amylolide; anticholinergic agents such as ipratropium, atropin or oxytropium; hormones such as cortisone, hydrocortisone or prednisolone; xanthin, eg Aminophyllin, cholineteophilinate, lysine theophylline or theophylline; and therapeutic proteins and peptides such as insulin or glucagon. Where appropriate, the drug may be in the form of a salt (eg, as an alkali metal or amine salt or as an acid addition salt) or an ester (eg, lower) to optimize the activity and / or stability of the drug. It will be apparent to those skilled in the art that it can be used as an alkyl ester) or as a solvate (eg, a hydrate).

Other exemplary therapeutic agents useful for combination therapy include, but are not limited to, the above agents, radiotherapy, hormone antagonists, hormones and their release factors, thyroid and antithyroid drugs, estrogen and progestin, androgen, Corticostimulatory hormones; corticosteroids and their synthetic analogs; inhibitors of the synthesis and action of corticohormones, insulin, oral hypoglycemic agents, and pancreatic endocrine pharmacological substances, calcification and bone turnover Affected agents: growth factors such as calcium, phosphoric acid, adrenal hormones, vitamin D, calcitonine, vitamins such as water-soluble vitamins, vitamin B complex, ascorbic acid, fat-soluble vitamins, vitamins A, K, and E, Hormones, chemokines, muscarinic receptor agonists and antagonists; anticholineresterase agents; drugs that act at neuromuscular junctions and / or autonomic ganglia; catecholamines, sympathomimetics, and adrenergic receptor agonists or antagonists; and 5- Included are hydroxytryptamine (5-HT, serotonin) receptor agonists and antagonists.

Therapeutic agents include histamine and histamine antagonists, bradikinin and brazikinin antagonists, 5-hydroxytryptamine (serotonin), lipids produced by biotransformation of the products of selective hydrolysis of membrane phospholipids, eicosanoids, prostaglandins, thromboxane, Leukotrienes, aspirins, non-steroidal anti-inflammatory agents, analgesic and antipyretic agents that inhibit the synthesis of prostaglandins and thromboxane, selective inhibitors of inducible cyclooxygenase, selective inhibitors of inducible cyclooxygenase-2, autacoids, parasecretory hormones , Somatostatin, gastrin, cytokines that mediate interactions involved in humoral and cellular immune responses, lipid-derived autacoids, eicosanoids, β-adrenergic agonists, ipratropium, glucocorticoids, methylxanetin, sodium channel blockers, Drugs for pain and inflammation, such as opioid receptor agonists, calcium channel blockers, membrane stabilizers and leukotriene inhibitors, may also be included.

Additional therapeutic agents that are intended for use in combination with compositions containing cinetylcarol include diuretics, vasopressin, agents that affect the retention of water by the kidneys, rennin, angiotensin, myocardial ischemia. Drugs useful in the treatment of, antihypertensive agents, angiotensin converting enzyme inhibitors, β-adrenalinergic receptor antagonists, agents for treating hypercholesterolemia, and agents for treating dyslipidemia. Can be done.

Other intended therapeutic agents include drugs used to control gastric acidity, drugs to treat digestive ulcers, drugs to treat gastroesophageal reflux disease, gastrointestinal motility improvers, Anti-vomiting drugs, drugs used for irritable bowel syndrome, drugs used for diarrhea, drugs used for constipation, drugs used for inflammatory bowel disease, used for bile duct disease Drugs and drugs used for pancreatic diseases can be mentioned. Therapeutic agents used to treat protozoal infections, drugs used to treat malaria, amebiasis, giardiasis, trichomoniasis, trypanosomiasis, and / or leishmaniasis, and / or worm disease Drugs used in chemotherapy. Other therapeutic agents include antibacterial agents, sulfonamides, trimethoprim-sulfamethoxazole quinolone, and agents for urinary tract infections, penicillins, cephalosporins, and others, β-lactam antibiotics, aminoglycosides. Antiviral agents, including drugs, protein synthesis inhibitors, tuberculosis, mycobacterium avium complex disease, and drugs used in chemotherapy for Hansen's disease, antifungal agents, non-retroviral agents and anti-retroviral agents.

Examples of therapeutic antibodies that can be combined with the subject compounds include, but are not limited to, anti-receptor tyrosine kinase antibodies (cetuximab, panitumumab, trastuzumab), anti-CD20 antibodies (rituximab, tocitumomab), and alemtuzumab, bevacizumab, And other antibodies such as gemtuzumab.

In addition, therapeutic agents used for immunomodulation, such as immunomodulators, immunosuppressants, tolerants, and immunostimulants, can be contemplated by the methods of the invention. In addition, therapeutic agents, hematopoietic agents, growth factors, inorganic substances, and vitamins, anticoagulants, thrombolytic agents, and antiplatelet agents that act on blood and hematopoietic organs can be contemplated by the methods of the invention.

Other therapeutic agents that can be combined with the compounds of interest, both of which are incorporated herein by reference in their entirety, Goodman and Gilman, The Pharmacological Basis of Therapeutics, 10th Edition, Hardman, Limbird and Gilman, Or you can find it in Physician's Desk Reference.

The compounds described herein, including cinetylcarol and Spiranthes sinensis extract, can be used in combination with one or more other agents, depending on the condition being treated. Therefore, in some embodiments, the compounds of the present disclosure are co-administered with other agents described above. When used in combination therapy, the compounds described herein can be administered simultaneously with or separately from the second agent. This combination administration comprises administering the two drugs simultaneously in the same dosage form, simultaneously in separate dosage forms, and separately. That is, the compounds described herein and any of the above agents can be formulated together in the same dosage form and administered simultaneously. Alternatively, the compounds of the present disclosure and any of the above agents can be administered simultaneously, with both agents present as separate formulations. In another alternative, the compounds of the present disclosure may be administered, followed immediately by any of the above agents, and vice versa. In separate dosing protocols, the compounds of the present disclosure and any of the above agents can be administered at intervals of minutes, hours, or days.

The amount of compound administered depends on the mammal being treated, the severity of the disorder or condition, the rate of administration, the pharmacokinetics of the compound and the discretion of the prescribing physician. However, effective doses can range from about 1 to about 100 mg / kg body weight per day. Effective doses are about 0.1-80 mg, about 0.5-80 mg, about 1-80 mg, about 10-80 mg, about 20-80 mg, about 50-80 mg, about 0.1-per kg of body weight per day. It can be in the range of 10 mg, about 0.5-10 mg, about 1-100 mg, about 10-100 mg, about 20-100 mg, or about 50-100 mg. As a non-limiting example, an effective dose can be from about 0.01 kg / kg body weight per day to 1.0 kg / kg body weight. As another non-limiting example, an effective dose can be about 0.6-5 mg / 60 kg per day in single or divided doses. For a 120 kg human, this would be an amount of about 1.2-10 mg per day.

Administration of the compounds of the present disclosure can be brought about by any method that allows delivery of the compounds to the site of action. Effective amounts of the compounds of the present disclosure, in either single or multiple doses, are intravenous, intraperitoneal, parenteral by rectal, buccal, intranasal and percutaneous, or intraarterial injections. Administration of acceptable agents of similar utility, intramuscular, subcutaneous, oral, topical, as an inhalant, or via an permeable or coating device such as a stent or arterial insertion type columnar polymer. It can be administered by any of the forms.
Administration

In some embodiments, the compositions of the present disclosure, including the dosage forms described herein, are intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal. It is formulated for one or more routes of administration by administration, rectal administration, intramuscular administration, subcutaneous administration, intraosseous administration, transmucosal administration, inhalation administration, and / or intraperitoneal administration. In some embodiments, the subject composition is administered by oral administration. In some embodiments, the subject composition is administered by intravenous injection. In some embodiments, the subject composition is administered by a combination of oral administration and intravenous injection. In some embodiments, the subject composition is formulated for topical administration.

The subject composition can be administered alone or in combination with one or more other agents that are also commonly administered in the form of pharmaceutical compositions. If desired, the subject compound and other agents (s) can be mixed into a preparation, both components can be formulated into separate preparations and combined and used separately or simultaneously. You can also do it.

Administration of the compounds or pharmaceutical compositions of the present disclosure can be brought about by any method that allows delivery of the compounds to the site of action. These methods include the oral route, the intraduodenal route, parenteral injection (including intravenous, intraarterial, subcutaneous, intramuscular, intravascular, intraperitoneal or infusion), topical (eg, transdermal application), intrarectal. These include administration, local delivery by catheter or stent, or inhalation. The compound can also be administered intrafat or intrathecally.

The compositions of the present disclosure can also be delivered by osmotic or coating devices such as stents or arterial insertion type columnar polymers. Such a method of administration can assist in the prevention or improvement of restenosis after procedures such as, for example, balloon angiogenesis. Without being bound by theory, the compounds of the present disclosure can slow or inhibit the migration and proliferation of smooth muscle cells in the arterial wall that contribute to restenosis. The compounds of the present disclosure can be administered, for example, by local delivery from a stent strut, from a stent graft, from a graft, or from a stent cover or sheath. In some embodiments, the compounds of the present disclosure are miscible with the matrix. Such a matrix can be a polymeric matrix and can help bind the compound to the stent. Suitable polymer matrices for such use include, for example, lactone-based polyesters or copolyesters such as polylactic acid, polycaprolactone glycolide, polyorthoesters, polyacid anhydrides, polyamino acids, polysaccharides, polyphosphazene, poly (ether). -Ester) copolymer (eg PEO-PLLA); polydimethylsiloxane, poly (ethylene-vinyl acetate), acrylate-based polymer or copolymer (eg polyhydroxyethylmethylmethacrylate, polyvinylpyrrolidinone), polytetra Fluorinated polymers such as fluoroethylene and cellulose esters can be mentioned. Suitable matrices may be non-degradable or may be degraded over time to release the compound (s). The compounds of the present disclosure can be applied to the surface of a stent by various methods such as dip / spin coating, spray coating, dip coating, and / or brush coating. The compound can be applied in a solvent, the solvent can be evaporated and a layer of compound can be formed on the stent. Alternatively, the compound can be placed within the body of the stent or graft, eg, within a microchannel or micropore. Once implanted, the compound diffuses out of the body of the stent and contacts the arterial wall. Such stents are prepared by immersing a stent made to contain such micropores or microchannels in a solution of the compounds of the present disclosure in a suitable solvent and then evaporating the solvent. Can be done. Excess drug on the surface of the stent can be removed by additional simple solvent cleaning. In yet another embodiment, the compounds of the present disclosure can be covalently linked to a stent or graft. Covalent linkers that are degraded in vivo and result in the release of the compounds of the present disclosure can be used. Any biounstable link, such as an ester link, an amide link or an anhydride link, can be used for such purposes. The compounds of the present disclosure can also be administered intravascularly through a balloon used during angiogenesis. To reduce restenosis, extravasation of the compounds by application of the formulations of the present disclosure to the pericardium or adventitia can also be performed.

The various stent devices that can be used as described are disclosed, for example, in the following references, all of which are incorporated herein by reference: US Pat. No. 5,451,233; US Pat. No. 5,040,548; US Pat. No. 5061273; US Pat. No. 5496346; US Pat. No. 5,292,331; US Pat. No. 5,674,278; US Pat. No. 3,657,744; US Pat. No. 4,739,762; US Pat. No. 5,195984; US Pat. No. 5,292,331; US Pat. No.; US Pat. No. 5,879,382; US Pat. No. 6,344,053.

Subject compositions include, for example, tablets, capsules, pills, microspheres, powders, sustained release formulations, solutions, suspensions and other forms suitable for oral administration, sterile solutions, suspensions or emulsions. It may be a form suitable for parenteral injection, a form suitable for topical administration such as an ointment or cream, or a form suitable for intrarectal administration such as a suppository. The composition may be in a unit dosage form suitable for a single dose of the correct dose. The composition may include conventional pharmaceutical carriers or excipients and compounds according to the present disclosure as active ingredients. In addition, the composition may include other drugs or pharmaceuticals, carriers, adjuvants and the like.

Preparations for parenteral administration generally include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solution, emulsions or suspensions, including saline and buffer media. Parenteral vehicles include sodium chloride solution, Ringer's glucose, dextrose and sodium chloride, Ringer's lactate solution, or non-volatile oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (eg, those based on Ringer's glucose) and the like. Preservatives and other additives such as antibacterial agents, antioxidants, chelating agents, and inert gases may also be present.

If the compound of the present disclosure is administered in a composition comprising one or more agents and the half-life of the agent is shorter than that of the compound of the present disclosure, then the agent and the compound dosage form of the present disclosure will be administered accordingly. Can be adjusted.

In one aspect, the disclosure provides a composition comprising an amount of cinetylcarol, which is present in an amount effective in reducing the inflammatory response. Administration of the disclosed compositions can reduce the inflammatory response, reduce the level of one or more free radicals in the inflammatory response, and / or reduce tissue swelling.

In some embodiments of the present disclosure, administration of the composition reduces the inflammatory response, iNOS, COX-2, TNF-alpha, PGE2, IL-6, IL-1β, IL-33, NLRP3, pERK, Reduced expression or activity of one or more biological markers such as NFκB, MMP2, and MMP9 is caused.

Inflammation is generally the body's reaction to invading infectious microorganisms, generally simply to increase blood flow to the affected area, release chemicals that elicit white blood cells, increase plasma flow, and remove debris. It results in the appearance of spheres (or astrocytes in the case of the brain). Anything that stimulates the inflammatory response can be said to be inflammatory. Reduction of inflammation can be measured by any suitable method, including the methods described herein. Reduction of inflammation can also be measured by reduced expression levels of the biological markers described herein. In general, the expression level of a biological marker can be achieved by measuring the level of expression of DNA, RNA, gene transcription, and / or protein. Examples of common techniques for measuring the expression levels of the biological markers described herein include PCR, qRT-PCR, microarrays, NanoString, RNA-seq, western blots, and spectroscopic measurements. .. In some embodiments, the composition has an inflammatory response of at least about 0.1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 60%, or about. It is effective in reducing 75% to 90% or more.

In some embodiments, administration of the composition reduces the level of one or more free radicals in the inflammatory response. The one or more free radicals decreases in response to administration of the composition, superoxide radical anion _{(O 2} ^-), hydrogen peroxide _{(H 2 O} 2), hydroxyl radical (.OH), singlet A group consisting of oxygen ( ¹ (O ₂ ), peroxynitrite (ONOO ⁻ ), hydrogen peroxide (NO), nitrosonium cation (NO ⁺ ), and nitroxyl anion (NO ⁻ ) can be mentioned.

The effect of the composition on reducing free radicals can be measured, for example, by nitrite quantification. In some embodiments, the composition has an inflammatory response of at least about 0.1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 60%, or about. It is effective in reducing 75% to 90% or more.

In some embodiments, administration of the composition reduces tissue swelling. Non-limiting examples of tissues in which swelling can be reduced include skin, epithelium, synovial tissue, tendons, cartilage, ligaments, bones, muscles, organs, hard membranes, blood vessels, bone marrow, and extracellular matrix.

Reduction of tissue swelling can be determined by changes in the swelling index. As a non-limiting example, the swelling index can be calculated by dividing the area of the animal's damaged limb by the area of the control animal's undamaged limb. The swelling index can be expressed as a ratio based on the swelling index of the injured limb as opposed to the injured limb. In some embodiments, the swelling index of the injured limb as opposed to the injured limb before and after said administration can be recorded for comparison. As another non-limiting example, inflammation in an animal model can be induced by injecting a complete Freund's adjuvant (CFA) into one paw. The swelling index can be calculated using the formula shown below and can be shown as a ratio based on the swelling index before injection (day 0). In some embodiments, the composition causes tissue swelling at least about 0.1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 60%, or about. It is effective in reducing 75% to 90% or more.

In some embodiments, administration of the composition has anti-liver fibrosis activity. Examples of anti-liver fibrosis include, but are not limited to, lipid accumulation in hepatic stellate cells, downregulation of hepatic stellate cell proliferation, inhibition of hepatic stellate cell mobility, prevention of hepatic stellate cell activation, And / or signs such as decreased synthesis of ECM protein.

In general, liver fibrosis is a scarring process in response to liver injury. In general, liver fibrosis is an over- or over-proliferative wound healing in which excess connective tissue is built in the liver. Extracellular matrix (ECM) is overproduced, poorly degraded, or a combination thereof. Over time, this process can lead to liver cirrhosis and can lead to severe complications, including portal hypertension, liver failure, and / or liver cancer. is there.

Scars are generally denser, thicker, and generally paler than the surrounding tissue due to insufficient blood supply to the scar, and scars are structural to the destroyed tissue. It replaces, but generally cannot perform all of the missing organizational functions. Scars are generally composed of collagen fibers, which often limit normal elasticity in the tissues involved. Reduction of scar tissue can be assessed by a population of cell types within the site of injury. For example, reduced formation of scar tissue can be measured by a simple measurement of scar width or area of scar tissue (Wilgus et al., 2003). In addition, a histological assessment can be made on the restoration of structural complexity within the healed tissue compared to normal tissue. In some embodiments, scar formation is at least about 0.1%, 2%, 5%, 10%, depending on the composition disclosed herein, such as a composition or dosage form comprising cinetylcarol. It can be reduced by 15%, 20%, 25%, 30%, 50%, 60%, or about 75% to 90% or more.
Pharmaceutical composition

In one aspect, the disclosure provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition is (a) an amount of Spiranthes sinensis extract (SSE) or cinetylcarol, which is present in an amount effective in reducing the inflammatory response, and (b) pharmaceutically acceptable. Contains the carrier to be used. The pharmaceutical compositions of the present disclosure generally include an active ingredient (eg, a compound of the present disclosure or a pharmaceutically acceptable salt and / or coordination compound thereof), and one or more pharmaceutically acceptable excipients. It contains forms, carriers including, but not limited to, inert solid diluents and fillers, diluents, sterile aqueous solutions and various organic solvents, penetration enhancers, solubilizers and adjuvants.

In some embodiments, the disclosure provides a pharmaceutical composition comprising a certain amount of Spiranthes sinensis extract and / or cinetylcarol formulated for administration to a subject in need thereof. In some embodiments, the pharmaceutical composition is about 0.001 to 1000 mg, 0.01 to 100 mg, 0.1 to 200 mg, 3 to 200 mg, 5 to 500 mg, 10 to 100 mg, 10 to 1000 mg, 50 to 200 mg. , Or 100-1000 mg of Spiranthes sinensis extract and / or cinetylcarol. In some embodiments, the pharmaceutical composition is about 0.001 μg or more than about 0.001 μg, about 0.01 μg or more than about 0.01 μg, about 0.1 or more than about 0.1, about 0.5 μg or More than about 0.5 μg, about 1 μg or more than about 1 μg, about 2 μg or more than about 2 μg, about 3 μg or more than about 3 μg, about 4 μg or more than about 4 μg, about 5 μg or more than about 5 μg, about 6 μg or more than about 6 μg, about 7 μg Or more than about 7 μg, about 8 μg or more than about 8 μg, about 9 μg or more than about 9 μg, about 10 μg or more than about 10 μg, about 15 μg or more than about 15 μg, about 20 μg or more than about 20 μg, about 25 μg or more than about 25 μg, about 50 μg or More than about 50 μg, about 75 μg or more than about 75 μg, about 100 μg or more than about 100 μg, about 200 μg or more than about 200 μg, about 500 μg or more than about 500 μg, about 1000 μg or more than about 1000 μg, or more than Spiranthes sinensis extract and / or Includes cinetylcarol. In some embodiments, the pharmaceutical composition comprises at least about 44 μg of Spiranthes sinensis extract. In some embodiments, the pharmaceutical composition comprises at least about 44 μg of cinetylcarol. In some embodiments, the pharmaceutical composition comprises an amount of Spiranthes sinensis extract in an amount of about 4.4-44 μg. In some embodiments, the pharmaceutical composition comprises an amount of about 4.4-44 μg of cinetylcarol. In some embodiments, the pharmaceutical composition comprises at least about 150 g of Spiranthes sinensis extract. In some embodiments, the pharmaceutical composition comprises at least about 250 g of cinetylcarol.

In some embodiments, the pharmaceutical composition is formulated for administration to a subject in need thereof. The compositions include intravenous administration, intraarterial administration, oral administration, parenteral administration, buccal administration, local administration, transdermal administration, rectal administration, intramuscular administration, subcutaneous administration, intraosseous administration, transmucosal administration, and inhalation administration. , And / or may be formulated for intraperitoneal administration. In some embodiments, the composition is formulated for topical administration.

In some embodiments, the pharmaceutical composition is formulated as a unit dose in liquid, gel, semi-liquid, or solid form. In some embodiments, the pharmaceutical composition is formulated as a topical cream.

In some embodiments, the pharmaceutical composition may be formulated as a food, such as a solid or semi-solid diet. In some embodiments, the composition may be formulated into a beverage. In some embodiments, the composition may be formulated in a dietary supplement.

In some embodiments, administration of the disclosed pharmaceutical composition to a subject in need thereof may reduce the inflammatory response and reduce the expression or activity of one or more biological markers. it can. Without being bound by any theory, one or more biological markers include iNOS, COX-2, TNF-alpha, PGE2, IL-6, IL-1β, IL-33, NLRP3, pERK, NFκB. , MMP2, and MMP9.

In some embodiments, the disclosed pharmaceutical composition can be administered to a subject in need thereof to reduce the level of one or more free radicals in the inflammatory response. Without being bound by any theory, the one or more free radicals decreases in response to administration of the composition, superoxide radical anion (O2 ^-), hydrogen peroxide (H 2 _{O 2),} hydroxyl A group consisting of radicals (.OH), monomorphic oxygen ( ¹ (O ₂ ), peroxynitrite (ONOO ⁻ ), nitrogen peroxide (NO), nitrosonium cations (NO ⁺ ), and nitroxyl anions (NO ⁻ ). Can be mentioned.

In some embodiments, administration of the compositions disclosed herein can reduce the inflammatory response and reduce tissue swelling. Non-limiting examples of reduction of tissue swelling include skin, epithelium, synovial tissue, tendons, cartilage, ligaments, bones, muscles, organs, hardeners, blood vessels, bone marrow, and extracellular matrix.

In some embodiments, the pharmaceutical composition may be formulated in unit doses in liquid, gel, semi-liquid, semi-solid, or solid form.

In some embodiments, the pharmaceutically acceptable carrier, excipient is selected from the group consisting of water, alcohol, glycerol, chitosan, alginic acid, chondroitin, vitamin E, mineral oil, and dimethyl sulfoxide (DMSO). ..
Pharmaceutical composition for topical (eg, transdermal) delivery

In some embodiments, the present disclosure provides pharmaceutical compositions for transdermal delivery that contain the compounds of the present disclosure and pharmaceutical excipients suitable for transdermal delivery. The composition may be in solid form, liquid form, gel form, semi-liquid form, or semi-solid form. In some embodiments, the composition further comprises a second agent.

The compositions of the present disclosure include gels, water-soluble jellies, creams, lotions, suspensions, foams, powders, slurrys, ointments, solutions, oils, pastes, suppositories, sprays, emulsions. , Physiological saline solution, solution based on dimethylsulfoxide (DMSO), etc., may be formulated in a solid, semi-solid, or liquid form preparation suitable for local or topical administration. In general, a dense carrier can provide a region where exposure to the active ingredient is sustained. In contrast, solution formulations can result in immediate exposure of the active ingredient to selected areas.

The pharmaceutical composition may also include suitable solid or gel phase carriers or excipients that are compounds that allow the permeation of therapeutic molecules or increased delivery beyond the skin's stratum corneum permeation barrier. There are many of these permeation-enhancing molecules known to be excellent as topical preparations in the art. Examples of such carriers and excipients are, but are not limited to, moisturizers (eg urea), glycols (eg propylene glycol), alcohols (eg ethanol), fatty acids (eg oleic acid), Surface active substances (eg, isopropyl myristate and sodium lauryl sulfate), pyrrolidone, glycerol monolaurate, sulfoxide, terpene (eg, menthol), amines, amides, alcohols, alkanols, water, calcium carbonate, calcium phosphate, various sugars, Polymers such as starch, cellulose derivatives, gelatin, and polyethylene glycol can be mentioned.

Formulations for topical administration include ointments, lotions, creams, gels (eg, poroxamer gels), drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily substrates, thickeners, etc. may be required or desired. The disclosed compositions include, for example, microfibers, polymers (eg, collagen), nanospheres, aerosols, lotions, creams, fabrics, plastics, tissue engineered scaffolds, matrix materials, tablets, implantable containers. As powders, oils, resins, wound dressings, beads, microbeads, slow-release beads, capsules, injections, IV drip, pump devices, silicone implants, or any bioengineered material. Can be administered.

Another exemplary formulation for use in the methods of the present disclosure uses a transdermal delivery device (“patch”). Such transdermal patches can be used to provide the compounds of the present disclosure in controlled amounts by continuous or discontinuous infusion with or without another agent. ..

The construction and use of transdermal patches for delivering pharmaceuticals is well known in the art. See, for example, US Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches can be constructed for continuous, pulsed, or on-demand delivery of the drug.
Pharmaceutical composition for oral administration

In some embodiments, the present disclosure provides pharmaceutical compositions for oral administration that contain the compounds of the present disclosure and pharmaceutical excipients suitable for oral administration. The composition may be in solid form, liquid form, gel form, semi-liquid form, or semi-solid form. In some embodiments, the composition further comprises a second agent.

The pharmaceutical compositions of the present disclosure suitable for oral administration each contain a predetermined amount of the active ingredient in a powder or granule, a solution, or a suspension in an aqueous or non-aqueous solution, an oil-in-water emulsion, or a water-in-oil emulsion. It may be present as a separate dosage form, such as a capsule, cashier, or tablet, or as a liquid or aerosol spray contained in a liquid. Such dosage forms can generally be prepared by any of the pharmaceutical methods, including the step of associating the active ingredient with the carrier. Generally, the composition is prepared by uniformly and closely mixing the active ingredient with a liquid carrier and / or a finely divided solid support, and then, if necessary, shaping the product to the desired appearance. For example, tablets can optionally be prepared by compression or molding with one or more accessory components. Compressed tablets are machine-appropriate, optionally shaped active ingredients such as, but not limited to, binders, lubricants, inert diluents, and / or surfactants or dispersants. It can be prepared by compressing into a free-flowing form such as powder or granules mixed with the agent. Molded tablets can be made by molding a mixture of powdered compounds moistened with an inert liquid diluent on a suitable machine.

The present disclosure further includes anhydrous pharmaceutical compositions and dosage forms containing the active ingredient, as water can facilitate the decomposition of some compounds. For example, in the pharmaceutical arts, water can be added as a means of simulating long-term storage to determine properties such as shelf life or stability of the formulation over time (eg, 5%). The anhydrous pharmaceutical compositions and dosage forms of the present disclosure can be prepared using anhydrous or low water content components and low water or low humidity conditions. The pharmaceutical compositions and dosage forms of the present disclosure containing lactose can be anhydrous if substantial contact with moisture and / or humidity during manufacture, packaging and / or storage is expected. Anhydrous pharmaceutical compositions can be prepared and stored so that they remain anhydrous. Therefore, the anhydrous composition can be packaged using known materials that prevent exposure to water and can therefore be included in the appropriate prescribed kit. Examples of suitable packaging include, but are not limited to, unit dose containers such as sealed foils, plastics, blister packaging, and strip packs.

The active ingredient can be combined with a pharmaceutical carrier to form a close admixture, depending on conventional pharmaceutical formulation techniques. The carrier can take a wide variety of forms depending on the form of the desired preparation for administration. In the preparation of compositions for oral dosage forms, any conventional pharmaceutical medium, such as liquid preparations for oral use (eg, suspensions, solutions, and elixirs) or, in the case of aerosols, water, glycols, Oils, alcohols, flavors, preservatives, colorants, etc. can be used as carriers; or in the case of oral solid preparations, starches, sugars, microcrystalline cellulose, diluents, granulators, Carriers such as lubricants, binders, and disintegrants can be used, and in some embodiments lactose is not used. For example, suitable carriers include powders, capsules, and tablets with solid oral preparations. If desired, the tablets can be coated by standard aqueous or non-aqueous techniques.

Suitable binders for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, natural and synthetic rubbers such as gelatin, arabic rubber, sodium alginate, Arginic acid, other alginates, tragant powder, guar gum, cellulose and derivatives thereof (eg, ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose, crystalline cellulose, and them. A mixture of.

Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein are, but are not limited to, starch, calcium carbonate (eg, granules or powders), crystalline cellulose, powder. Examples include cellulose, dextrate, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof.

Disintegrants can be used in the compositions of the present disclosure to result in tablets that disintegrate when exposed to an aqueous environment. Excessive amounts of disintegrant can result in tablets that can disintegrate in the bottle. In excess, inadequate disintegration can occur and the rate and extent of release of the active ingredient (s) from the dosage form can vary. Dosage forms of the compounds disclosed herein can be formed using sufficient amounts of disintegrants that are not too little and not too much to alter the release of the active ingredient (s). The amount of disintegrant used may vary based on the type of formulation and mode of administration and may be readily identifiable to those of skill in the art. About 0.5 to about 15 weight percent disintegrant, or about 1 to about 5 weight percent disintegrant, can be used in the pharmaceutical composition. Disintegrants that can be used to form the pharmaceutical compositions and dosage forms of the present disclosure include, but are not limited to, agar, alginic acid, calcium carbonate, crystalline cellulose, croscarmellose sodium, crospovidone, poracrylin. Examples include potassium, sodium starch glycolate, potato starch or tapioca starch, other starches, pregelatinized starches, other starches, clays, other argins, other cellulosic, rubbers or mixtures thereof.

Lubricating agents that can be used to form the pharmaceutical compositions and dosage forms of the present disclosure include, but are not limited to, calcium stearate, magnesium stearate, mineral oils, light oils, glycerin, sorbitol, mannitol, polyethylene glycol. , Other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (eg peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, lauric acid Includes ethyl, agar, or mixtures thereof. Additional lubricants include, for example, thyroid silica gel, solidified aerosols of synthetic silica, or mixtures thereof. The lubricant can optionally be added in an amount of less than about 1 weight percent of the pharmaceutical composition.

If an aqueous suspension and / or glycyl agent is desired for oral administration, the active ingredients therein are various sweeteners or flavors, colorants or pigments, and if desired emulsifiers and /. Alternatively, suspending agents can be combined with diluents such as water, ethanol, propylene glycol, glycerin and various combinations thereof.

The tablets may be uncoated and may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a long-lasting effect. For example, time delay materials such as glycerin monostearate or glyceryl distearate can be used. Formulations for oral use include hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or the active ingredient in a water or oil medium such as peanut oil, fluidized. It can also be provided as a soft gelatin capsule mixed with paraffin or olive oil.

Surfactants that can be used to form the pharmaceutical compositions and dosage forms of the present disclosure include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. .. That is, a mixture of hydrophilic surfactants can be used, a mixture of lipophilic surfactants can be used, and a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant can be used. It can also be used.

The HLB value of a suitable hydrophilic surfactant can generally be at least 10, and the HLB value of a suitable lipophilic surfactant can generally be about 10 or less. An empirical parameter used to characterize the relative hydrophilicity and hydrophobicity of nonionic amphipathic compounds is the hydrophilic-lipophilic equilibrium (“HLB” value). Surfactants with lower HLB values have higher lipophilicity or hydrophobicity and higher solubility in oil, and surfactants with higher HLB values have higher hydrophilicity and higher solubility in aqueous solutions. Hydrophilic surfactants are generally considered to be compounds with an HLB value greater than about 10 and anionic, cationic, or amphoteric ionic compounds to which the HLB scale is generally not applicable. Similarly, lipophilic (ie, hydrophobic) surfactants are compounds with an HLB value equal to or less than about 10. However, the HLB value of surfactants is only a rough guideline commonly used to enable the formulation of industrial, pharmaceutical and cosmetic emulsions.

The hydrophilic surfactant may be ionic or nonionic. Suitable ionic surfactants include, but are not limited to, alkylammonium salts; fusidates; fatty acid derivatives of amino acids, oligopeptides, and polypeptides; glyceride derivatives of amino acids, oligopeptides, and polypeptides; lysithin and Lysolecithin hydride; lysolecithin and lysolecithin hydride; phospholipids and their derivatives; lysopholipids and their derivatives; carnitine fatty acid ester salts; alkylsulfate salts; fatty acid salts; docusate sodium; acylactylates; monoglycerides and diglycerides Examples include monoacetylated tartrate and diacetylated tartrate; succinylated monoglycerides and diglycerides; citrates of monoglycerides and diglycerides; and mixtures thereof.

Within the above group, examples of ionic surface active substances include: lecithin, lysolecithin, phospholipids, lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; alkylsulfate salts; fatty acid salts; sodium docusate; Acylactylates; monoglyceride and diglyceride monoacetylated tartrate and diacetylated tartrate; succinylated monoglyceride and diglyceride; monoglyceride and diglyceride citrate; and mixtures thereof.

Ionic surfactants include lecithin, lysolecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylate, phosphatidylserine, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, lysophosphatidylserine, PEG-phosphatidylserine. Amin, PVP-phosphatidylethanolamine, fatty acid lactylate, stearoyl-2-lactilate, stearoyllactilate, succinylated monoglyceride, mono / diglyceride mono / diacetylated tartrate, mono / diglyceride citrate, chorylsarcosine, Caproic acid, capric acid, capric acid, laurate, myristic acid, palmitic acid, oleic acid, lysinolic acid, linoleic acid, linolenic acid, stearic acid, lauryl sulfate, terracecil sulfate, docusate, lauroyl carnitine, palmitoyl carnitine, myristyl carnitine , And their salts and mixtures may be in ionized form.

Hydrophilic nonionic surface active substances include, but are not limited to, alkyl glucosides; alkyl maltosides; alkyl thioglucosides; lauryl macrogol glycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; poly such as polyethylene glycol alkyl phenols. Oxyalkylene alkylphenol; polyoxyalkylene alkylphenol fatty acid ester such as polyethylene glycol fatty acid monoester and polyethylene glycol fatty acid diester; polyethylene glycol glycerol fatty acid ester; polyglycerin fatty acid ester; polyoxyalkylene sorbitan fatty acid ester such as polyethylene glycol sorbitan fatty acid ester; Hydrophilic ester exchange reaction product with at least one member of the group consisting of, glycerides, vegetable oils, hardened vegetable oils, fatty acids, and sterols; polyoxyethylene sterols, derivatives, and analogs thereof; polyoxyethylated vitamins and theirs. Derivatives; polyoxyethylene-polyoxypropylene block copolymers; and mixtures thereof; polyethylene glycol sorbitan fatty acid ester and hydrophilic ester exchange reaction formation with at least one member of the group consisting of polyols and triglycerides, vegetable oils, and cured vegetable oils. You can list things. The polyol may be glycerol, ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol, or saccharides.

Other hydrophilic nonionic surface active substances include, without limitation, PEG-10 lauric acid, PEG-12 lauric acid, PEG-20 lauric acid, PEG-32 lauric acid, PEG-32 dilauric acid, PEG-. 12 oleic acid, PEG-15 oleic acid, PEG-20 oleic acid, PEG-20 dioleic acid, PEG-32 oleic acid, PEG-200 oleic acid, PEG-400 oleic acid, PEG-15 stearic acid, PEG-32 distearate Acid, PEG-40 stearic acid, PEG-100 stearic acid, PEG-20 dilauric acid, PEG-25 glyceryl trioleic acid, PEG-23 dioleic acid, PEG-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG- 20 glyceryl stearate, PEG-20 glyceryl oleate, PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryl laurate, PEG-40 palm kernel oil, PEG-50 cured castor oil, PEG-40 Sunflower oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 cured castor oil, PEG-60 cured castor oil, PEG-60 corn oil, PEG-6 capric acid / capricyl glyceride, PEG-8 capric acid / Caprylyl glyceride, polyglyceryl-10 lauric acid, PEG-30 cholesterol, PEG-25 phytosterol, PEG-30 soysterol, PEG-20 trioleic acid, PEG-40 sorbitan oleate, PEG-80 sorbitan laurate, polysorbate 20 , Polysolvate 80, POE-9 lauryl ether, POE-23 lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearyl ether, tocopheryl PEG-100 succinic acid, PEG-24 cholesterol, polyglyceryl-10 olein Acids, Tween 40, Tween 60, sucrose monostearate, sucrose monolaurate, sucrose monopalmitate, PEG10-100 nonylphenol series, PEG15-100 octylphenol series, and poroxamar.

Suitable lipophilic surfactants are simply examples: fatty alcohol; glycerol fatty acid ester; acetylated glycerol fatty acid ester; lower alcohol fatty acid ester; propylene glycol fatty acid ester; sorbitan fatty acid ester; polyethylene glycol sorbitan fatty acid ester; sterol and sterol. Derivatives; polyoxyethylated sterols and sterol derivatives; polyethylene glycol alkyl ethers; sugar esters; sugar ethers; lactic acid derivatives of monoglycerides and diglycerides; polyols and at least one member of the group consisting of glycerides, vegetable oils, hardened vegetable oils, fatty acids and sterols. Hydrophilic ester exchange reaction products with; oil-soluble vitamins / vitamin derivatives; and mixtures thereof. In this group, preferred lipophilic surfactants include glycerol fatty acid esters, propylene glycol fatty acid esters, and mixtures thereof, or preferred lipophilic surfactants are polyols and vegetable oils, hardened vegetable oils, And a hydrophobic transesterification product with at least one member of the group consisting of triglycerides.

In one embodiment, the composition may include a solubilizer to ensure good solubilization and / or dissolution of the compounds of the present disclosure and to minimize precipitation of the compounds of the present disclosure. .. This can be particularly important for compositions for parenteral use, such as compositions for injection. Solubilizers may also be added to increase the solubility of hydrophilic drugs and / or other constituents such as surfactants, or to maintain the composition as a stable or uniform solution or dispersion. Can be done.

Examples of suitable solubilizers include, but are not limited to: alcohols and polyols such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediol and its isomers, glycerol. , Pentaerythritol, sorbitol, mannitol, transctor, dimethylisosorbide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydroxypropylmethylcellulose and other cellulose derivatives, cyclodextrin and cyclodextrin derivatives, etc .; polyethylene with an average molecular weight of about 200 to about 6000. Glycol ethers such as tetrahydrofurfuryl alcohol PEG ether (glycoflor) or methoxyPEG; amide and other nitrogen-containing compounds such as 2-pyrrolidone, 2-piperidone, epsilon-caprolactam, N-alkylpyrrolidone, N-Hydroxyalkylpyrrolidone, N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide and polyvinylpyrrolidone, etc .; esters such as ethyl propionate, tributyl citrate, triethyl acetyl citrate, tributyl acetyl citrate, triethyl citrate, Ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycol monoacetate, propylene glycol diacetate, ε-caprolactone and its isomers, δ-valerolactone and its isomers, β-butyrolactone and its isomers; Other solubilizers known in the art, such as dimethylacetamide, dimethylisosorbide, N-methylpyrrolidone, monooctanoin, diethylene glycol monoethyl ether, and water.

A mixture of solubilizers can also be used. Examples include, but are not limited to, triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropylcyclodextrin, Examples include ethanol, polyethylene glycol 200-100, glycoflor, transctor, propylene glycol, and dimethylisosorbide. Particularly preferred solubilizers include sorbitol, glycerol, triacetin, ethyl alcohol, PEG-400, glycoflor and propylene glycol.

The amount of the solubilizer that can be included is not particularly limited. The amount of a given solubilizer can be limited to bioacceptable amounts, which can be readily determined by one of ordinary skill in the art. In some cases, conventional techniques such as distillation or evaporation are used before bringing the composition to the patient, including, for example, solubilizers in amounts well in excess of the bioacceptable amount to maximize the concentration of the drug. It may be advantageous to remove excess solubilizer. If present, the weight ratio of the solubilizer may be 10%, 25%, 50%, 100%, or up to about 200% by weight, based on the combined weight of the drug and other excipients. .. If desired, very small amounts of solubilizers such as 5%, 2%, 1% or even lower% can be used. In general, the solubilizer may be present in an amount of about 1% to about 100% by weight, more generally from about 5% to about 25%.

The composition may further comprise one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, but are not limited to, tackifiers, antifoaming agents, buffers, polymers, antioxidants, preservatives, chelating agents, viscoelasticity regulators, isotonicization. Agents, flavors, colorants, odorants, opalescent agents, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.

In addition, acids or bases can be incorporated into the composition to facilitate processing, enhance stability, or for other reasons. Examples of pharmaceutically acceptable bases include amino acids, amino acid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate, magnesium hydroxide, aluminum magnesium silicate, Examples thereof include synthetic aluminum silicate, synthetic hydrocalcite, magnesium hydroxide, diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine, triethylamine, triisopropanolamine, trimethylamine, tris (hydroxymethyl) aminomethane (TRIS) and the like. Acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acid, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acid, formic acid, fumaric acid, gluconic acid, hydroquinonesulfonic acid, isoascorbic acid, Pharmaceuticals such as lactic acid, maleic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartrate acid, thioglycolic acid, toluenesulfonic acid, uric acid, etc. A base, which is an acceptable acid salt, is also suitable. Salts of polybasic acids such as sodium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate can also be used. If the base is a salt, the cation can be any convenient pharmaceutically acceptable cation, such as ammonium, alkali metals, alkaline earth metals and the like. Examples include, but are not limited to, sodium, potassium, lithium, magnesium, calcium and ammonium.

Suitable acids are pharmaceutically acceptable organic or inorganic acids. Examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid and the like. Examples of suitable organic acids are acetic acid, acrylic acid, adipic acid, alginic acid, alcan sulfonic acid, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid. , Hydroquinone sulfonic acid, isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, Examples thereof include thioglycolic acid, toluene sulfonic acid and uric acid.
Pharmaceutical composition for injection

In some embodiments, the present disclosure provides injectable pharmaceutical compositions containing the compounds of the present disclosure and pharmaceutical excipients suitable for injection. The constituents and amounts of the drug in the composition are as described herein.

The novel compositions of the present disclosure for administration by injection include aqueous or oily suspensions, or emulsions with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs. Included are mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical vehicles.

Saline aqueous solutions are also customarily used for injection. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol and the like (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils can also be used. Reasonable fluidity can be maintained, for example, by using a coating such as lecithin to maintain the required particle size in the case of dispersion and by using surfactants. Prevention of microbial action can be provided by various antibacterial and antifungal agents such as parabens, chlorobutanols, phenols, sorbic acid, thimerosal and the like.

Sterile injections are prepared by incorporating the required amount of the compounds of the present disclosure together with the various other components listed above in a suitable solvent and, if necessary, subsequent filtration sterilization. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing the basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for preparing sterile injections, certain desirable preparation methods are to obtain powder of the active ingredient and any additional desired ingredients from the solution which has been sterile filtered in advance, vacuum drying and It is a freeze-drying technique.
Pharmaceutical composition for inhalation

Compositions for inhalation or blowing include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, and powders. The liquid or solid composition may contain the appropriate pharmaceutically acceptable excipients described above. The composition is preferably administered by the oral or nasal respiratory route for local or systemic effects. The composition, which is preferably in a pharmaceutically acceptable solvent, can be atomized by using an inert gas. The atomized solution can also be inhaled directly from the spray device or the spray device can be attached to a face mask tent or intermittent positive pressure ventilator. The solution, suspension, or powder composition can preferably be administered orally or nasally from a device that delivers the formulation in a suitable manner.
Other pharmaceutical compositions

The pharmaceutical composition is suitable for cinetylcarol or Spiranthes sinensis extract and for sublingual, buccal, rectal, intraosseous, intraocular, intranasal, epidural, or intraspinal administration. It can also be prepared to include the compositions described herein, such as compositions containing one or more pharmaceutically acceptable excipients. Excipients and their use in pharmaceutical compositions are well known in the art. For example, Anderson, Philip O .; Knoben, James E .; Troutman, William G, Handbook of Clinical Drug Data, 10th Edition, McGraw-Hill, 2002, all of which are incorporated herein by reference in their entirety. Pratt and Taylor, Principles of Drug Action, 3rd Edition, Churchill Livingston, New York, 1990; Katzung, Basic and Clinical Pharmacology, 9th Edition, McGraw Hill, 20037ybg; Goodman and Gilman, The Pharmacological Basis of See Therapeutics, 10th Edition, McGraw Hill, 2001; Remingtons Pharmaceutical Sciences, 20th Edition, Lippincott Williams & Wilkins., 2000; Martindale, The Extra Pharmacopoeia, 32nd Edition (The Pharmaceutical Press, London, 1999). I want to be.

In some embodiments, the compositions and methods administer one or more additional agents (eg, one, two, three, four, five, or more) separately or simultaneously. Including additional steps to do. Additional agents may include those useful in wound healing. Non-limiting examples of additional drugs include antibiotics (eg, aminoglycoside, cephalosporin, chloramphenicol, clindamycin, erythromycin, fluoroquinolone, macrolide, azolide, metronidazole, penicillin, tetracycline, trimetoprim). -Sulfamethotrexate, vancomycin), steroids (eg andlan (eg testosterone), cholesterol (eg cholesterol), colic acids (eg coric acid), corticosteroids (eg dexamethasone), estraene (eg dexamethasone) Estradiol), prednisone (eg progesterone), drug-induced and non-drug analgesics (eg morphine, codeine, heroine, hydromorphon, levorphanol, meperidine, methadone, oxidone, propoxyphene, fentanyl, methadone, naloxone) , Buprenorfin, butorphanol, nalbufin, pentazosin), chemotherapy (eg, anticancer drugs, eg, but not limited to, altretamine, asparaginase, bleomycin, busulfane, carboplatin, carmustin, chlorambusyl, cisplatin, cladribine, cyclophosphamide , Citalabine, dacarbazine, diethylstilvesterol, ethynyl estradiol, etopocid, floxuridine, fludalabine, fluorouracil, flutamide, goseleline, hydroxyurea, idarubicin, ifosfamide, leuprolide, levamisole, romustine, mechloretamine, medroxyl gesterone Melfaran, mercaptopurine, methotrexate, mitomycin, mittan, mitoxanthrone, paclitaxel, pentastatin, pipobroman, plicamycin, prednisone, procarbazine, streptozocin, tamoxiphene, teniposide, vinblastin, vincristine, etc., anti-inflammatory agents (eg, alcrophenac) Alcromethadone dipropionate; argastone acetonide; alpha amylase; amcinafal; amcinafide; amphenac sodium; amyprirose hydrochloride; anakinla; anilolac; anitrazafene; apazone; balsaladisotheno; Vincristine hydrochloride; bromeline; bropellamol; prednisone; calprofen; cyclop Lophen; Cintazone; Criprofen; Clobetazol propionate; Clobetazone butyrate; Piroxicam; Croticazone propionate; Colmethasone acetate; Coltodoxone; Decanoic acid; Defrazacoat; Delatestril; Depot testosterone; Desonide; Desoxymethasone; Dexamethacin Dipropionate; diclofenac potassium; diclofenac sodium; diflorazone diacetate; diflumidone sodium; diflunisal; diflupredonate; diphthalone; dimethylsulfoxide; drosinonide; endolison; enlimomab; enolicam sodium; epilyzole; etodrac; etofenamate; fervinac Phenamoll; Fembufen; Fenclofenac; Fenchlorac; Fendsar; Fempipalone; Fentiazak; Frazaron; Fluazacort; Flufenamic acid; Flumisol; Flunisolide acetate; Frunixin; Flunicinmeglumin; Fluocortinbutyl; Fluorometron acetate; Fruquazone; Fluletofen; fluticazone propionate; flaprofen; flobphen; halcinonide; halobetasol propionate; halopredon acetate; ibfenac; ibprofen; ibprofen aluminum; ibprofen piconol; ironidap; indomethacin; indomethacin sodium; indomethacin; indomethacin Sole; Intrazol; Isoflupredon acetate; Isoxepac; Isoxycam; Ketoprofen; Lofemizol hydrochloride; Piroxicam; Rotepredonor etabonate; Sodium meclofenamic acid; Mecrophenamic acid; Mechlorison dibutyrate; Mephenamic acid; Mesalamine; Mesecrazone; Mesterolone; Methandrostenolone; metenolone; metenolone acetate; methylprednisolone sleptate; morniflumate; nabumeton; nandrolone; naproxen; naproxen sodium; naproxol; nimazone; orsalazine sodium; orgotane; orpanoxin; oxandrolane; oxandrolane; Fenbutazone; Oxymethron; Paraniline hydrochloride; Sodium pentosanpoly sulfate; Fenbutazone sodium glycerate; Pilphenidone; Piroxicam; Piroxicam cinnate; Piroxicam aurami Pyrprofen; prednazart; preferon; prodolic acid; procazone; proxazole; proxazole citrate; rimexolone; romazarit; sarcorex; salnacedin; salsalate; sanguinarium chloride; secrazone; cermastamine; stanozolol; sudoxycam; sulindac; Tarniflumart; talosalate; tebuferon; tenidap; tenidap sodium; tenoxycam; tesicum; tesimid; testosterone; testosterone mixture; tetridamine; thiopinac; thixocortor pivalate; tolmethin; tolmethin sodium; trichronide; triflumidate; Pyraksodium), or antihistamines (eg, ethanolamine (such as diphenhydramine carbinoxamine), ethylenediamine (such as trypereneamine pyriramine), alkylamines (such as chlorpheniramine, dexchlorpheniramine, brompheniramine, triprolidine)) , Other antihistamines such as astemizole, loratadine, fexofenadine, brompheniramine, cremastine, acetaminophen, pseudoephedrine, triprolysine).
food

In one aspect, the disclosure provides a food composition comprising a food carrier and an amount of cinetylcarol. The food composition may be in a dosage form for administering cinetylcarol, such as the dosage form described herein. In some embodiments, the food composition comprises cinetylcarol at about 0.001 to 1000 mg, 0.01 to 100 mg, 0.1 to 200 mg, 3 to 200 mg, 5 to 500 mg, 10 to 100 mg, 10 to 10. Included in cinetylcarol between 1000 mg, 50-200 mg, or 100-1000 mg. In some embodiments, the food composition is about 0.001 μg or more than about 0.001 μg, about 0.01 μg or more than about 0.01 μg, about 0.1 or more than about 0.1, about 0.5 μg or More than about 0.5 μg, about 1 μg or more than about 1 μg, about 2 μg or more than about 2 μg, about 3 μg or more than about 3 μg, about 4 μg or more than about 4 μg, about 5 μg or more than about 5 μg, about 6 μg or more than about 6 μg, about 7 μg Or more than about 7 μg, about 8 μg or more than about 8 μg, about 9 μg or more than about 9 μg, about 10 μg or more than about 10 μg, about 15 μg or more than about 15 μg, about 20 μg or more than about 20 μg, about 25 μg or more than about 25 μg, about 50 μg or Includes cinetylcarols greater than about 50 μg, greater than about 75 μg or greater than about 75 μg, greater than about 100 μg or greater than about 100 μg, greater than about 200 μg or greater than about 200 μg, greater than about 500 μg or greater than about 500 μg, greater than about 1000 μg or greater than about 1000 μg, or greater. In some embodiments, the food composition comprises at least about 4.4 μg of cinetylcarol. In some embodiments, the food composition comprises an amount between about 4.4 and 44 μg of cinetylcarol. In some embodiments, the food composition comprises at least about 44 μg of cinetylcarol. In some embodiments, the food composition comprises a Spiranthes sinensis extract.

The packaging of the disclosed food compositions can be achieved by any of a variety of suitable methods. For example, the food composition can be packaged as a beverage, solid food, and / or semi-solid food. In some cases, the disclosed food composition is packaged as a food product, such as one or more of the group consisting of snack bars, cereal products, bakery products, and dairy products.

In some embodiments, the disclosed pharmaceutical composition is packaged in a food formulation formulated as a unit dose. Without being bound by any limitation, unit doses are formulated as beverages, dietary supplements, solid foods, and / or semi-solid foods.
treatment

In some embodiments, the present disclosure provides pharmaceutical compositions and methods that reduce free radicals, reduce inflammatory responses, and / or promote wound healing. In some embodiments, the method comprises administering to a subject in need thereof a dosage form containing cinetylcarol, which is formulated for administration to the subject. In some embodiments, the dosage form further comprises a pharmaceutical carrier. In some embodiments, the dosage form comprises a Spiranthes sinensis extract. The method may include the step of administering any of the compositions disclosed herein, such as dosage forms, pharmaceutical compositions, or food compositions. Administration of the composition and measurement of its effect are described herein, for example with respect to the various compositions disclosed herein.

As mentioned above, the present disclosure provides methods for reducing the inflammatory response, including the step of administering a dosage form containing cinetylcarol to a subject in need thereof. The composition may be any of the cinetylcarol-containing compositions described herein. In one embodiment, the dosage form of cinetylcarol is formulated for administration to a subject. In a related embodiment, the dosage form is a pharmaceutical composition comprising a pharmaceutically acceptable carrier, examples of which are described herein. In a further related embodiment, the dosage form comprises a Spiranthes sinensis extract, such as the extracts described herein.

In some embodiments, the present disclosure provides pharmaceutical compositions and methods for treating inflammation, including but not limited to liver fibrosis. The disclosed compositions can be used to provide anti-inflammatory, anti-liver fibrosis, and / or antioxidant activity. In some embodiments, the composition comprises an effective amount of cinetylcarol. In some embodiments, the pharmaceutical composition comprises an effective amount of Spiranthes sinensis extract. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In some embodiments, the methods provided in the present invention can reduce inflammation in a subject. Inflammation is generally the body's reaction to invading infectious microorganisms, generally to increase blood flow to the affected area, release chemicals that elicit white blood cells, increase plasma flow, and remove debris. It results in the appearance of monocytes (or astrocytes in the case of the brain). The inflammatory stimulant can be anything that stimulates the inflammatory response. In some embodiments, in addition to reducing inflammation in subjects in response to tissue damage, the compositions and methods provided include, for example, asthma, eczema, sinusitis, atherosclerosis, rheumatoid arthritis, inflammation. It can also be used to treat disorders associated with pathologically elevated levels of inflammatory cells, including enteral diseases, cutaneous and systemic mastocytosis, asthma, and multiple sclerosis. Treatments with the provided polypeptides can be used, for example, to reduce pruritus in wounds during healing. In general, pruritus causes the release of histamine by mast cells. In some embodiments, the compositions provided are used to reduce mast cell degranulation and histamine release. In some embodiments, it is provided to treat conditions with histamine release, including, but not limited to, pruritus, scratches, sinus irritation, allergic cough, red eyes, asthma, and eczema. Use the composition.

In some embodiments, the inflammatory response is associated with chronic or acute immune impairment. In some embodiments, the composition is a disease or condition associated with a chronic or acute immunodeficiency, or an immunodeficiency such as a primary immunodeficiency and a secondary or acquired immunodeficiency, and / or autoimmunity. Provide a method for treating a disorder. Examples of autoimmune disorders include lupus, scleroderma, certain types of hemolytic anemia, vasculitis, type I diabetes, Graves' disease, rheumatoid arthritis, multiple sclerosis, Goodpasture syndrome, pernicious anemia, and some. Types include myopthay, and Lime's disease (late stage). Illustrative primary immunodeficiency disorders include severe combined immunodeficiency disease (SCID), DiGeorgia syndrome, hyper-IgE syndrome (also known as Job syndrome), unclassifiable immunodeficiency disease (CVID), and chronic granulomatous disease. Disease (CGD), Wiskott-Aldrich Syndrome (WAS), Autoimmune Lymphoproliferative Syndrome (ALPS), High IgM Syndrome, Leukocyte Adhesion Deficiency (LAD), NF-κB Essential Modifier (NEMO) Mutation, Selection Immunoglobulin A deficiency, X-linked agammaglobulinemia (XLA, also known as Breton-type agammaglobulinemia), and X-linked lymphoproliferative disorder (XLP) and ataxia-capillary dilatation. .. An example of secondary immunodeficiency is AIDS. Other immune disorders include allergies such as seasonal allergies, mastocytosis, perennial allergies, anaphylaxis, food allergies, allergic rhinitis, and atopic dermatitis. Additional non-limiting examples include acute diffuse encephalomyelitis (ADEM), Addison's disease, antiphospholipid antibody syndrome (APS), malregenerative anemia, autoimmune hepatitis, Celiac's disease, Crohn's disease, true Diabetes (Type 1), Good Pasture Syndrome, Graves Disease, Guillain-Barré Syndrome (GBS), Hae (OMS), Ophthalitis, Eau de Thyroiditis, Oemphigus, Polyarthritis, Primary Biliary Liver Cirrhosis, Psoriasis, rheumatoid shimato's disease, erythematosus, polysclerosis, severe myasthenia, ocular clonus-myokronus syndrome, arthritis, Reiter syndrome, hyperan arteritis, temporal arteritis ("giant cell arteritis") Also known as "arteritis"), warm autoimmune hemolytic anemia, Wegener's granulomatosis, generalized alopecia, Shagas' disease, chronic fatigue syndrome, autonomic neuropathy, endometriosis, purulent sweat adenitis, Included are qualitative cystitis, neuromuscular tonicity, sarcoidosis, giant cell arteritis, ulcerative colitis, leukoplakia, and genital pain. Other disorders include impaired bone reabsorption and thrombosis.

In some embodiments of the present disclosure, administration of the composition reduces one or more symptoms associated with an autoimmune disease, a neurodegenerative disease, or a disease associated with inflammation. Non-limiting examples of such symptoms include inflammation, fatigue, dizziness, malaise, hyperthermia and hyperthermia, extreme sensitivity to cold hands and feet, muscle and joint weakness and rigidity, weight changes, etc. Depending on digestive or gastrointestinal problems, hypotension or hypertension, irritability, anxiety, or depression, infertility or diminished sexual drive (hypotension), changes in blood glucose, and the type of autoimmune disease, the organ or Examples include increased tissue size or destruction of organs or tissues.

In some embodiments, administration of the composition comprising cinetylcarol is not limited to, but is limited to, reducing joint swelling, lowering serum anti-collagen levels, and / or bone resorption, cartilage damage, pannus, and. / Or it is effective in improving the symptoms associated with rheumatoid arthritis, including reduction of joint pathology such as inflammation. In some embodiments, the methods of the subject cause inflammation of joints (eg, legs or ankles) at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%. It is effective in reducing by 60%, or about 75% to 90%. In some embodiments, inflammation is reduced by at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 60%, or about 75% -90% or more. To do. In some embodiments, the methods of the subject have serum antiserum type II collagen levels of at least about 10%, 12%, 15%, 20%, 24%, 25%, 30%, 35%, 50%, 60. It is effective in reducing%, 75%, 80%, 86%, 87%, or about 90% or more. In some embodiments, the method of the subject has an ankle histopathological examination score of about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%. , 80%, 90% or more. In some embodiments, the method of the subject has a knee histopathological examination score of about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%. , 80%, 90% or more.

In some embodiments, it is effective to administer a composition comprising cinetylcarol to treat asthma. The compounds or pharmaceutical compositions described herein can also be used to treat toxicemia and sepsis. In one embodiment, the compounds or pharmaceutical compositions described herein are used to treat rheumatoid arthritis (RA). In yet another embodiment, the compounds or pharmaceutical compositions described herein are used to treat contact dermatitis or atopic dermatitis. Examples of contact dermatitis include irritant dermatitis, phototoxic dermatitis, allergic dermatitis, photoallergic dermatitis, contact urticaria, and systemic contact dermatitis. Irritant dermatitis can occur when the substance is overused on the skin when the skin is highly sensitive to certain substances. Atopic dermatitis, sometimes referred to as eczema, is a type of dermatitis, atopic dermatitis.

Reduction of inflammation can be measured using a variety of methods, such as those described herein. In some embodiments, reduced inflammatory response is a sign of reduced inflammation. Methods for measuring reduction in inflammatory response include, for example, from iNOS, COX-2, TNF-alpha, PGE2, IL-6, IL-β, IL-33, NLRP3, pERK, NFκB, MMP2, and MMP9. The expression or activity of one or more biological markers selected from the group is reduced.

Reduction of inflammation can also be measured, for example, by reducing the density of inflammatory cell types such as monocytes or astrocytes. Reduction of inflammation can be measured, for example, by reducing the density of inflammatory cell types such as neutrophils, mast cells, basophils, and monocytes. Reduction of inflammation can be calculated by in vivo measurement of neutrophil activity. In addition, factors such as the frequency of mast cell degranulation or measurements of histamine levels or reactive oxygen species levels can be used as criteria for reducing inflammation. The level of inflammation refers to the transcriptional levels of specific genes, eg, interleukin-alpha, interleukin-beta and interleukin-gamma, tumor necrosis factor-alpha, interleukin 1β, interleukin 2, interleukin 4, interleukin 5, interleukin Indirectly by investigating genes such as leukin 6, interleukin 8, interleukin 12, interleukin 18, interleukin 23, interleukin 27, CD4, CD28, CD80, CD86, MHCII, and iNOS by qRT-PCR. It can also be measured. Reduction of inflammation can be measured by measuring pro-inflammatory cytokine levels in tissues and / or body fluids of interest, including plasma. The mechanism of ACT peptide action is inhibition of inflammatory cell migration and / or pro-inflammatory chemicals (histamine, active oxygen species), and interleukin (IL) -1, IL-6, IL-8 and tumor necrosis factor. It should be noted that this may be due to inhibition of pro-inflammatory cytokines such as (TNF).

In some embodiments, the reduction in inflammatory response is also demonstrated by reduced levels of one or more free radicals associated with the inflammatory response, such as reactive oxygen species or reactive nitrogen species. Reactive oxygen species (“ROS”) and reactive nitrogen species (“RNS”) are two major types of compounds that have important physiological functions in an organism. ROS and RNS include not only radicals, but also other related non-radical species formed during the intracellular oxidation process. These free radicals and related species are also involved, among other things, in the regulation of signal transduction from membrane receptors, immunological and inflammatory responses, smooth muscle relaxation, redox homeostasis, apoptosis, and vascular tone.

The ROS, superoxide radical anions ^- and radical species such as hydroxyl radical _{(.OH) (O 2.)} ; And, peroxides (ROOR), hydrogen peroxide _{(H 2 O} 2), singlet oxygen ⁽¹ Examples include related non-radical species such as O ₂ ) and peroxynitrite (ONOO ⁻ ). RNS includes radical species such as nitric oxide (.NO); and related non-radical species such as nitrosonium cation (NO ⁺ ), nitroxyl anion (NO ⁻ ), and peroxynitrite (ONOO ⁻ ). Be done. Numerous enzymatic processes lead to the formation of ROS (Fink MP., "Reactive oxygen", including reactions catalyzed by xanthine oxidase, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoforms and cyclooxygenase isoforms. species as mediators of organ dysfunction caused by sepsis, acute respiratory distress syndrome, or hemorrhagic shock: potential benefits of resuscitation with Ringer's ethyl pyruvate solution, Curr Opin Clin Nutr Metab Care, 2002, Vol. 5, pp. 167-174). RNS is an intermediate that can be formed as a result of the oxidation of L-arginine. This oxidation process is catalyzed by nitric oxide synthase (NOS) isoforms, resulting in the formation of nitric oxide (.NO). There are three NOS isoforms: neuron NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). These isoforms are expressed in a variety of intracellular locations and tissues, and non-limiting examples thereof include cardiomyocytes, glial cells, skeletal cells, neutrophils, vascular smooth muscle cells, and platelets. Nitric oxide includes nitrosonium cations (NO ⁺ ), nitroxyl anions (NO ⁻ ), and peroxynitrite (ONOO ⁻ , a toxic compound formed by the reaction of nitric oxide with superoxide anions). Can be converted to non-radical RNS.

ROS and RNS can also play a decisive role as mediators of a number of different intracellular signaling cascades, including inflammatory processes (Droge W, "Free radicals in the physiological control of cell function", Physiol Rev., 82. Volume (No. 1): pp. 47-95, 2002). In particular, ROS is an NF-κB (Pantano et al., “Redox-sensitive kinases of the nuclear factor-kappaB signaling pathway”, Antioxid Redox Signal., Vol. 8 (No. 9-10): 1791- 806, 2006; Gloire et al., "NF-kappaB activation by reactive oxygen species: fifteen years later", Biochem Pharmacol, Vol. 72: 1493-1505, 2006) and activator protein (AP) -1 (Liu). Et al., "Redox-dependent transcriptional regulation", Circ Res 97: 967-974, 2005; Lin et al., "Superoxide dismutase inhibits the expression of vascular cell adhesion molecule-1 and intracellular cell adhesion molecule-1 induced by tumor necrosis". Factor-alpha in human endothelial cells through the JNK / p38 pathways ”, Arterioscler Thromb Vasc Biol, Vol. 25: pp. 334-340, 2005) has been implicated in the regulation of signal transduction. Redox-dependent signaling mechanisms are also associated with activation of proinflammatory mitogen-activated protein kinases (MAPKs), including p38 and JNK (Nagai et al., "Pathophysiological roles of ASK1-MAP kinase signaling pathways". , J Biochem Mol Biol, Vol. 40: pp. 1-6, 2007). The expression of nitric oxide synthase isoforms has been associated with inflammation, and the induction of inducible nitric oxide synthase (iNOS) is lipocytokine (LPS), endotoxin, or IFN-γ, TNF-αc, and. It can be initiated by inflammatory cytokines such as IL-1 (Guziket et al., "Nitric oxide and superoxide in inflammation and immune regulation", J Physiol Pharmacol, Vol. 54 (4): 469-87, 2003).

Appropriate regulation of ROS and RNS provides protection against oxidative stress and also provides important mediators in cellular processes. However, overproduction or improper clearance of ROS and RNS can cause damage to cellular constructs such as proteins, DNA, and membrane lipids; dysfunction of intracellular signaling cascades; cytotoxicity; and enzyme inactivation. Occurs. Free radicals range from various forms of cancer, including type 2 true diabetes, atherosclerosis, chronic inflammatory conditions, ischemic / reperfusion injury, sepsis and some neurodegenerative diseases. It has been implicated in the development of pathology in disease and pathological processes (Droge W. Free radicals in the physiological control of cell function. Physiol Rev, 2002, Vol. 82: pp. 47-95).

In some embodiments, administration of the compositions according to the present disclosure raises the level of one or more free radicals at least about 0.1%, 2%, 5%, 10%, 15%, 20%, 25%. , 30%, 50%, 60%, or about 75% to 90% or more effective. The decrease in the level of one or more free radicals can be measured by any suitable assay, for example by nitrite quantification. Decreased levels of one or more free radicals can also be measured in assays for antioxidant activity, such as the assays disclosed herein.

The inflammatory response also causes tissue swelling. As described herein, swelling is generally the body's response to injury as a result of increased migration of fluid and leukocytes to the area of inflammation. In general, swelling is an abnormal enlargement of any body part. Swelling can be due to fluids, including blood, bony dysplasia, muscles, or many other things. In some cases, the swelling can be edema, which represents fluid or swelling that has accumulated in the tissues outside the joint capsule, including swelling of the calf or thigh. In some cases, the swelling can be exudate, which represents what the fluid is inside the joint capsule, such as a swollen ankle or knee. In some cases, swelling can be arthremia, a condition in which blood is present in the exudate within the joint capsule, indicating either ligament injury such as ACL rupture or fracture. This is generally determined by extracting some fluid from the joint capsule with a needle. Arthritis can be acute or chronic. Acute arthritis refers to swelling that occurs within 24 hours of injury. If the swelling occurs within the first 2 hours, it is probably associated with arthritis and should be confirmed by a medical professional. Chronic arthritis refers to long-term swelling that can be difficult to detect but can be very detrimental if left untreated.

In some embodiments, a reduction in the inflammatory response is demonstrated by a reduction in tissue swelling. As described herein, reduction of swelling is found in tissues including skin, epithelium, synovial tissue, tendons, cartilage, ligaments, bones, muscles, organs, hardeners, blood vessels, bone marrow, and extracellular matrix. be able to. Reduction of tissue swelling can be determined by changes in the swelling index. As a non-limiting example, the swelling index can be calculated by dividing the area of the animal's damaged limb by the area of the control animal's undamaged limb. The swelling index can be expressed as a ratio based on the swelling index of the injured limb as opposed to the injured limb. In some embodiments, the swelling index of the injured limb as opposed to the injured limb before and after said administration can be recorded for comparison. As another non-limiting example, inflammation in an animal model can be induced by injecting a complete Freund's adjuvant (CFA) into one paw. In some embodiments, administration of the composition causes tissue swelling at least about 0.1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 60%, Or it is effective in reducing about 75% to 90% or more.

In one aspect, the disclosure provides pharmaceutical compositions and methods for promoting wound healing, comprising the step of administering a dosage form containing cinetylcarol to a subject in need thereof. The composition may be any of the cinetylcarol-containing compositions described herein. In one embodiment, the dosage form of cinetylcarol is formulated for administration to a subject. In a related embodiment, the dosage form is a pharmaceutical composition comprising a pharmaceutically acceptable carrier, examples of which are described herein. In a further related embodiment, the dosage form comprises a Spiranthes sinensis extract, such as the extracts described herein. In some embodiments, the promotion of wound healing is such that the rate of wound healing is compared to the rate of healing of the corresponding untreated wound or to the rate of pretreatment of the treated wound. Including increasing. Improvements in the rate of wound healing increase the rate of healing by 10%, 20%, 30%, 40%, 50%, 100%, 150%, 200%, 250%, 500%, 1000% or more. Including.

In some embodiments, the promotion of wound healing is compared to fibrosis in which fibrosis occurs in the corresponding untreated wound, or in the partially healed portion of the wound during pretreatment healing. Including reducing. Fibrosis is a pathological process that involves scar formation by connective tissue and overproduction of extracellular matrix in response to tissue damage. The repair process generally involves two distinct phases: the damaged cells are replaced by cells of the same type, leaving no evidence of damage, the regenerative phase; and the connective tissue replaces normal parenchymal tissue. With a phase known as fibrosis or fibrosis. This repair process is beneficial at first, but becomes pathogenic if not properly controlled, resulting in a substantial deposition of extracellular matrix components in which normal tissue is replaced with permanent scar tissue. Occurs. In some embodiments, administration of the compositions of the present disclosure results in fibrosis of about 10% or more than about 10%, about 20% or more than about 20%, about 30% or more than about 30%, about 40. % Or more than about 40%, about 50% or more than about 50%, about 100% or more than about 100%, about 150% or more than about 150%, about 200% or more than about 200%, about 250% or about 250% Super, about 500% or more than about 500%, about 1000% or more than about 1000%, or more reduction.

In some embodiments, the methods provided in the present invention can reduce scar tissue formation in a subject after tissue injury. Scar tissue can replace damaged areas of internal organs such as damaged skin and underlying muscles, damaged myocardium, or liver. Scars are dense, thick, usually paler than surrounding tissue due to inadequate blood supply, and scars are structural replacements for destroyed tissue, although In general, it is not possible to perform all of the missing organizational functions. Scars are generally composed of collagen fibers, which often limit normal elasticity in the tissues involved. Therefore, if scar tissue limits the range of muscle movement or affects the lymphatic or circulatory system, it interferes with proper fluid circulation. Post-injury glio scar tissue to the brain or spinal cord is one of the major obstacles to the recovery of post-injury neural function to the central nervous system. Reduction of scar tissue can be assessed by a population of cell types within the site of injury. For example, a decrease in glue scar tissue can be estimated by an increase in the ratio of neurons to astrocyte cells. Reduction of scar formation can be measured by a simple measurement of scar width or area of scar tissue (Wilgus et al., "Reduction of scar formation in full-thickness wounds with topical celecoxib treatment", Wound Rep Reg Vol. 11: 25-34 pages, 2003). In addition, a histological assessment can be made on the restoration of structural complexity within the healed tissue compared to normal tissue. In some embodiments, scar formation is at least about 0.1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 60%, or about 75% to. It can be reduced by 90% or more.

The decrease in fibrous tissue formation in a subject can be measured by the physician's clinical judgment to assess whether the normal structure and function of a given tissue and / or organ in the subject has been restored after the procedure. As an example, for psoriasis, a medical professional will evaluate the subject's skin to determine if the patch of reddish skin covered with scaly white ridges has diminished. Certain types of psoriasis are characterized by a comedone-like appearance (pustular psoriasis) or a burn appearance (erythroderma). In such cases, the medical professional will determine if the procedure resulted in a reduction in these symptoms. Clinic pathologists and / or training in the case of subjects for which biopsy material is clinically available and / or necessary by medical professionals, or tissues or organs in animal models of human disease. The histological pathologist prepares a tissue fragment of the biopsy material and evaluates the histological structure to determine whether reduction of fibrosis and restoration of normal tissue structure and function have occurred. The area of fibrosis relative to normal tissue can also be evaluated quantitatively in such histological preparations.

Providing wound healing involves treating a subject undergoing cosmetic surgery or having tissue damage. In other embodiments, the present disclosure provides methods for treating damage and / or promoting tissue regeneration using the subject composition. Examples of tissue injuries include scratches, cuts, lacerations, crush wounds, compression wounds, extension injuries, bites, scratches, shots, explosions, decubitus, body piercing, punctures, heat wounds, gun wounds, wind wounds, sunburn , Chemical burns, surgical wounds, surgical interventions, medical interventions, host rejection after cell, tissue or organ transplantation, drug action, drug side effects, decubitus, radiation damage, cosmetic skin wounds, visceral damage, disease processes (eg , Asthma, cancer), infection, infectious agents, developmental processes, maturation processes (eg, acne), genetic abnormalities, developmental abnormalities, environmental toxins, allergens, scalp injuries, facial injuries, jaw injuries, foot injuries, toe injuries , Finger injury, bone injury, genital injury, joint injury, excretory injury, ocular trauma, corneal injury, muscle injury, adipose tissue injury, lung injury, tracheal injury, hernia, anal injury, hemorrhoids, ear injury, retinal injury, skin Injury, abdominal injury, arm injury, lower limb injury, ligament injury, back injury, birth injury, premature birth injury, poison bite, sting, tendon injury, ligament injury, heart injury, heart valve injury, vasculature injury, cartilage injury, Lymphatic system injury, cranial brain injury, dislocation, esophageal perforation, fistula, nail injury, foreign body, fracture, frost injury, hand injury, heat stress injury, rupture, cervical injury, self-injury, shock, traumatic soft tissue injury, spinal cord injury , Spinal cord injury, contusion, contusion, tendon injury, ligament injury, cartilage injury, chest injury, tooth injury, trauma, nervous system injury, aging, aneurysm, stroke, gastrointestinal injury, infarction, ischemic injury, fracture, contusion , Contusion, stroke, infarction, aneurysm, hernia formation, ischemia, fistula, dislocation, radiation, cells, tissue or organ transplantation, or cancer. In some embodiments, the compounds described herein are used to treat and / or prevent acne.

In some embodiments, promoting wound healing involves restoring to the wound tissue some amount of normal tissue mechanical properties, such as tensile strength after tissue damage in the subject. "Tensile strength" refers to the amount of stress or strain required to destroy a tissue or wound. Within 1 month, 2 months, 3 months, 4 months, 5 months or 6 months after treatment, the tensile strength of treated wounds is 60%, 65% of the tensile strength of undamaged tissue. , 70%, 75%, 80%, 85%, 90%, 95%, 100%. In some embodiments, the mechanical properties of the tissue are restored, including increasing the tensile strength of the healed injury in the subject until it approaches or reaches the tensile strength of the normally undamaged tissue. The method comprises the step of administering to the subject one or more of the compositions provided in the present invention (eg, cinetylcarol or Spiranthes sinensis extract) in a pharmaceutically acceptable carrier. A method is provided.

Examples of tissues that can be treated to restore tensile strength using the disclosed compositions or methods include musculoskeletal structures / tissues and damage to the skin covering these structures. For example, the provided compositions can be administered to improve the tensile strength of articulated joints, bones, cartilage, tendons, or ligaments. Improvements in tensile strength can include improving the tensile strength of the skin under high stress / strain forces, such as the skin covering the elbows, knees, or feet. The most common problem associated with healing joint injuries is that excessive scarring of these areas leads to contraction and non-extension of the healed joint areas. This leads to serious cosmetic and psychological consequences. The properties of the composition can be used to regulate and reduce the formation of such scar tissue, thereby increasing joint mobility.

In some embodiments, promoting wound healing involves increased tissue regeneration of tissue damage in the subject. In contrast to scarring, tissue regeneration involves the restoration of tissue to its original structural, functional, and physiological condition. This is also referred to herein as the "complexity" of the organization. Recovery is partial or complete, ie 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100 compared to native or control levels. % Recovery, or any amount of recovery in between. As an example, in the case of skin injury, tissue regeneration may involve recovery of hair follicles, glandular structures, blood vessels, muscles, or fat. In the case of brain injury, tissue regeneration can involve maintenance or recovery of neurons. As an example, in the case of skin, improvement in tissue regeneration can be assessed by measuring the volume of fibrous scar tissue relative to normal regenerated skin as a ratio. As another example, it is possible to count on separate regenerating structures, such as regenerating skin glands, standardized for the volume of the wound area.

In one embodiment, tissue regeneration involves the recruitment and differentiation of stem cells to replace damaged cells. The primary role of stem cells in living organisms is to maintain and repair the tissues in which they are found. Stem cell differentiation refers to the process by which unspecialized cells (eg, stem cells) acquire the characteristics of specialized cells such as skin cells, nerve cells, heart cells, liver cells, or muscle cells. As an example, in the case of skin injury, tissue regeneration can involve the differentiation of stem cells present in the epithelium into hair follicles (Alonso and Fuchs, 2003). In the case of brain injury, tissue regeneration can involve the differentiation of stem cells into neurons. The methods provided can enhance stem cell differentiation after tissue damage in a subject. Enhanced stem cell differentiation marks endogenous or planted stem cells and determines the frequency of differentiation and incorporation of marked stem cells into normal tissue structure, clinically acceptable genetic or It can be measured by providing other means. As another example, it is known that certain structures, such as hair follicles, are regenerated from endogenous stem cells after tissue damage. As such, standardized hair follicle counts for tissue-damaged areas serve as a quantitative assessment of enhanced stem cell differentiation.

Subsequent examples are provided for purposes of illustrating various embodiments of the invention and are not meant to limit the invention in any manner. This example, along with the methods described herein, is a representative preferred embodiment of the present, is an example, and is not intended to be a limitation of the scope of the invention. Modifications and other uses in the embodiments that are within the spirit of the invention as defined by the claims are also envisioned by those skilled in the art.
(Example 1)
How to purify cinetylcarol

The whole air-dried plant of Spiranthes sinensis (PERS.) AMES was purchased from the local herbarium, Hualien, Taiwan in November 2013, and the Herbarium of the Department of Taiwan, National Taiwan, National Taiwan, Taiwan, Taiwan, Taiwan, Taiwan, Taiwan, Taiwan. No., collected on April 12, 1934), identified by comparison with specimens of evidence already deposited.

Spiranthes sinensis air-dried whole plants (eg 2.4 kg) were pulverized and extracted directly with EtOAc (eg 50 L) 3 times at room temperature for 72 hours each and the combined extracts concentrated under reduced pressure. A crude extract (eg, 160 g) of n-hexane- EtOAc-methanol (1: 0: 0, 10: 1: 0, 5: 1: 0, 3: 1: 0, 1: 1: 0, 0: 1, respectively) : 0, 0:40: 1, 0:30: 1, 0:20: 1 and 0:10: 1) were subjected to column chromatography on silica using a gradient mixture as an eluate. Thirteen fractions were obtained from the TLC results. The fourth fraction was further chromatographed on a silica gel column using n-hexane- EtOAc (3: 1) as the eluate to give 10 fractions. Fractions 4-4 and 5 were found to have crystalline appearance after filtration and were purified by recrystallization to obtain cinetylcarol. Compound identity was fully characterized by comparing their spectral data (IR, NMR and mass) with the information reported in the literature.

Preserved compositions were made for subsequent experiments by dissolving the extract in DMSO at a concentration of 1 mg / mL based on the total volume of DMSO.
(Example 2)
Cytotoxicity of Spiranthes sinensis and cinetylcarol Method Cell culture

The RAW264.7 mouse (murine) macrophage cell line was purchased from the Biorelocation Collection and Research Center (BCRC, Taiwan). Cells were supplemented with 100 U / mL penicillin, 100 μg / mL sodium pyruvate and 10% fetal bovine serum (FBS; Gibco Inc.) in Dalveco modified eagle medium (DMEM, Gibco Inc., NY, USA) at 37 ° C., 5 Incubated with% CO ₂ .

Cells were also treated with 0.01% DMSO as vehicle control. Cells were seeded in 12-well plates for 24 hours at a density of 6 × 10 ⁵ cells / well, then incubated in serum-free medium containing 0.1% BSA for 3 hours with various concentrations of Spiranthes sinensis extract or SI. It was treated and then incubated in the presence of LPS (1 μg / mL) for 4 hours.
Cell viability

To examine the biocompatibility of the extracts of the compositions of the present disclosure, MTT assay (3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Invitrogen), ratio. An assay based on color analysis) was performed.

Briefly, ³ cells 8x10 per well were seeded on 96-well plates and incubated overnight at 5% CO ₂ , 37 ° C. Cells were treated with various concentrations (5, 20, 50 and 100 μg / mL) of Spiranthes sinensis extract for 24 hours, after incubation, 20 μL (5 mg / mL) of MTT solution per well was added and incubated for an additional 4 hours. .. The medium was removed and formazan was dissolved by adding 100 μl / well of DMSO (Sigma-Aldrich) and OD was measured at 570 nm using a microplate reader (ELISA reader, Thermo Labsystems). The percentage of living cells was estimated by comparing with untreated control cells. The average absorbance value of the control cells was defined as 100% survival rate.

The data are mean ± S. Represented as E. A statistical comparison of the results was made using one-way analysis of variance (ANOVA). The mean in each column followed by different letters is significantly different at p <0.05 by Turkey's test.
result

Cytotoxicity of Spiranthes sinensis extract and cinetylcarol in RAW264.7 cells analyzed by MTT assay. Cell viability was unaffected by treatment with Spiranthes sinensis extract (5, 20, 50 and 100 μg / mL) and cinetylcarol (0.1, 1, 5 and 10 μM) at 24 hours (FIG. 1). ). These observations suggested that Spiranthes sinensis and cinetylcarol are not toxic to cells.
(Example 3)
Effect of Spiranthes sinensis extract and cinetylcarol on CFA-induced inflammation in mice Methods Animals

C57BL / 6 mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan) and kept in controlled environmental conditions at room temperature (22 ± 2 ° C.) and humidity (60 ± 10%). A 12-hour light (6 am-6 pm) and dark cycle was maintained throughout the study. Animal studies were approved by the National Dong-Hwa University Animal Ethics Committee and used according to the National Dong-Hwa University "Guide for the Care and Use of Laboratory Animals". All animals that underwent CFA developed severe inflammation typically in the hind limbs, were very red with an inflammation score of 200% swelling, and developed edema within 20 minutes. Experiments: CFA group: This group was only induced by CFA and was not treated, Spiranthes sinensis extract (SS) group: This group has various concentrations (0.5, 0.3 and 0.06 mg / kg) b.w) received an intraperitoneal injection of the Spiranthes sinensis extract; cinetylcarol (SI) group: this group received intraperitoneal SI at varying concentrations (4.4, 2.2 and 0.44 μg / kg). Received an intraperitoneal injection; triamcinolone (TS): this group received an intraperitoneal injection of 0.3 mg / kg triamcinolone (clinical steroid) for positive control; sebatrim (SE): this group received 3 mg / kg Intraperitoneal injection of triamcinolone (clinical NSAID) was received as a positive control. There were 5 mice in each group. To produce a chronic inflammatory response, mice were subcutaneously injected with 20 μL of complete Freund's adjuvant (CFA, Mycobacterium tuberculosis, Sigma, St. Louis, MO, USA) into the sole of the right hind paw (i.pl.). .. The left hind paw was used as a control.

After induction of CFA (day 0), mice were treated with SS extract and SI by intraperitoneal injection up to day 2 three times. The anti-inflammatory effect was examined for 4 days and the swelling index was recorded daily. The swelling index was calculated by the following formula and expressed as a ratio based on the swelling index before injection (day 0).

The data are mean ± S. Represented as E. A statistical comparison of the results was made using a one-way dispersion analysis (ANOVA). The mean within each column followed by different letters was significantly different at p <0.05 by Tukey's test.
result

This effect was determined in CFA-induced inflamed mice to evaluate the anti-inflammatory properties of the Spiranthes sinensis extract and SI. All treatment groups had anti-inflammatory effects when compared to the CFA group. High concentrations of Spiranthes sinensis extract between treatments had a better anti-inflammatory effect. The Spiranthes sinensis extract and the positive control group, especially the steroid group, had a marked recovery of swelling 3 days after discontinuation of treatment (Fig. 2). However, the highest concentration of 0.5 mg / kg of Spiranthes sinensis extract resulted in less foot swelling recovery than that of 0.3 mg / kg triamsinolone. The SI group had an even better anti-inflammatory effect than that of the Spiranthes sinensis extract. It had less recovery from foot swelling than that of 0.3 mg / kg triamcinolone. However, a SI of 0.44 μg / kg was a better effect than that of a SI of 4.4 μg / kg, indicating that high concentrations of SI may have resulted in excessive suppression of immune factors. (Fig. 3).
(Example 4)
Effect of CFA-induced inflammation on blood MMP2 and MMP9 in mice of Spiranthes sinensis extract Method Animals

C57BL / 6 mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan) and kept in controlled environmental conditions at room temperature (22 ± 2 ° C.) and humidity (60 ± 10%). A 12-hour light (6 am-6 pm) and dark cycle was maintained throughout the study. Animal studies were approved by the National Dong-Hwa University Animal Ethics Committee and used according to the National Dong-Hwa University "Guide for the Care and Use of Laboratory Animals". All animals that underwent CFA developed severe inflammation typically in the hind limbs, were very red with an inflammation score of 200% swelling, and developed edema within 20 minutes. Experiments: CFA group: This group was only induced by CFA and was not treated, Spiranthes sinensis extract (SS) group: This group has various concentrations (0.5, 0.3 and 0.06 mg / kg) Intraperitoneal injection of spiranthes sinensis extract from b.w); Cinetylcarol (SI) group: Intraperitoneal injection of SI at various concentrations (4.4, 2.2 and 0.44 μg / kg); Triamcinolone: Intraperitoneal injection of triamcinolone, a 0.3 mg / kg clinical steroid for positive controls; Sebatrim: Intraperitoneal injection of 3 mg / kg clinical NSAID, triamcinolone, for positive controls. There were 5 mice in each group.
Gelatin zymography

The activity of MMP2 and MMP9 was assayed by gelatin zymography because this method can detect the active and latent forms of MMP2 and MMP9 based on their molecular weight. Blood was collected from CFA-induced mice on day 4 and centrifuged to obtain serum. Serum was collected and quantified using a Bradford dye (Bio-Rad). An 8% SDS-PAGE gel containing 10% gelatin was prepared. A 7.5 μg protein sample was loaded onto the gel and electrophoretic separation was performed at 80 V for 2-3 hours. Incubated in color buffer at 37 ° C. for 16 hours. Finally, the gel was stained in 0.1% Coomassie blue R-250 (Bio-Rad) for 4 hours and then decolorized with fixed buffer. Gels were scanned using an Epson scanner and quantified using multi-gauge software (Fujifilm).

Samples derived from mice in the SS group were treated in the same manner. Blood samples were collected from the tail vein on day 4. Blood was preheated at 55 ° C. with 2 × load dye (0.125M Tris-HCl, pH 6.8, 4% SDS, 0.04% bromophenol blue, 20% glycerol). An 8% SDS-PAGE gel containing 10% gelatin was prepared. A 5 μL blood sample was loaded onto the gel and electrophoretic separation was performed at 80 V for 2-3 hours. After electrophoresis, the gel was washed twice with 50 mL of 2.5% Triton X-100 per gel, then in color buffer (0.05M Tris-HCl, pH 8.8, 5 mM CaCl ₂ , 0.02% NaN). Incubated in ₃ ) at 37 ° C. for 16 hours. Finally, the gel was stained in 0.1% Coomassie blue R-250 (Bio-Rad) for 4 hours and then decolorized with fixed buffer (45% methanol, 10% acetic acid). Gels were scanned using an Epson scanner and quantified using multi-gauge software (Fujifilm). This method can detect the active and latent forms of MMP9 by their exact molecular weight size.

Data were expressed as mean ± SEM. A statistical comparison of the results was made using one-way analysis of variance (ANOVA). The mean within each column followed by different letters was significantly different at p <0.05 by Tukey's test.
result

Zymography was applied to measure the effect of SI on blood MMP2 and MMP9 in CFA-induced inflamed mice. CFA-induced inflamed mice had higher MMP2 and MMP9 expression in the blood (Fig. 4). MMP quantification revealed that MMP9 levels were reduced in SI-treated groups at various concentrations in CFA-induced mice. SI of 0.44 μg / kg had an inhibitory effect on the activity of MMP9, but was not so remarkable at the MMP2 level. The results for SS samples are illustrated in FIG. 26, indicating that the Spiranthes sinensis extract has an inhibitory effect on the activity of MMP9, a type of gelatinase that is upregulated during liver fibrosis.
(Example 5)
Effect of Spiranthes sinensis extract and cinetylcarol on LPS-induced IL-1β, IL-6, iNOs and TNF-α mRNA expression in RAW264.7 cells Method Cell culture

The RAW264.7 mouse (murine) macrophage cell line was purchased from the Biorelocation Collection and Research Center (BCRC, Taiwan). Cells were supplemented with 100 U / mL penicillin, 100 μg / mL sodium pyruvate and 10% fetal bovine serum (FBS; Gibco Inc.) in Dalveco modified eagle medium (DMEM, Gibco Inc., NY, USA) at 37 ° C., 5 Incubated with% CO ₂ .

Cells were also treated with 0.01% DMSO as vehicle control. Cells were seeded in 12-well plates for 24 hours at a density of 6 × 10 ⁵ cells / well, then incubated in serum-free medium containing 0.1% BSA for 3 hours with various concentrations of Spiranthes sinensis extract or SI. It was treated and then incubated in the presence of LPS (1 μg / mL) for 4 hours.
Real-time PCR

Total RNA was prepared from freshly recovered RAW264.7 cells and isolated using trizol extraction. RNA samples were frozen at −80 ° C. until analysis. The cDNA was then prepared using the M-MLV RT kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Expression of selected cytokines and related genes in mouse tissues exposed to this Spiranthes sinensis extract and RAW264.7 cells was determined by SensiFAST SYBR No-ROX Kit (BIORINE, London, UK) as previously described. The PCR cycle conditions for TNF-alpha included the first 2 minutes of 95 ° C. heating step per run. The tube was then annealed at 95 ° C. for 5 seconds, annealed at 60 ° C. for 10 seconds and stretched at 72 ° C. for 20 seconds for 40 cycles. PCR conditions for iNOs, IL6 and IL1 included the first 2 minutes of 95 ° C. heating step per run. The tube was then annealed at 95 ° C. for 5 seconds, annealed at 64 ° C. for 10 seconds and stretched at 72 ° C. for 20 seconds for 38 cycles. In all cases, optical data was collected during the annealing phase. An internal standard with β-actin for quantifying the expression exhibited by each of the PCR products. The primers used in the real-time PCR of this example are listed in Table 1.

The data are mean ± S. Represented as E. A statistical comparison of the results was made using one-way analysis of variance (ANOVA). The mean within each column followed by different letters was significantly different at p <0.05 by Tukey's test.
result

We then investigated the effects of Spiranthes sinensis extracts (FIG. 5) and SI (FIG. 6) on LPS-induced IL-1β, IL-6 and IL-33 release. The results showed that the Spiranthes sinensis extract reduced LPS-induced inflammatory cytokines IL-1β and IL-6 transcription in a dose-dependent manner. We found that TNF-α, NLRP3 and iNOs mRNA levels were also significantly reduced by a 50 μg / mL Spiranthes sinensis extract in a similar manner. 5 μM SI had a better inhibition of inflammatory cytokine transcription. RAW264.7 cells were found in a dose-dependent manner after Spiranthes sinensis extract or SI treatment. An alternative illustration of the results for the Spiranthes sinensis extract is provided in FIG.
(Example 6)
Effect of COX-2 expression, NFκB and phosphor ERK activation on LPS-induced RAW264.7 cells of Spiranthes sinensis extract and cinetylcarol Method Cell culture

The RAW264.7 mouse (murine) macrophage cell line was purchased from the Biorelocation Collection and Research Center (BCRC, Taiwan). Cells were supplemented with 100 U / mL penicillin, 100 μg / mL sodium pyruvate and 10% fetal bovine serum (FBS; Gibco Inc.) in Dalveco modified eagle medium (DMEM, Gibco Inc., NY, USA) at 37 ° C., 5 Incubated with% CO ₂ .

Cells were also treated with 0.01% DMSO as vehicle control. Cells were seeded in 12-well plates for 24 hours at a density of 6 × 10 ⁵ cells / well, then incubated in serum-free medium containing 0.1% BSA for 3 hours with various concentrations of Spiranthes sinensis extract or SI. It was treated and then incubated in the presence of LPS (1 μg / mL) for 4 hours.
Western blot

The concentration of protein from whole cell solubilized products was determined by the Bradford assay. Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. The blot was then blocked overnight with 5% (wt / vol) defatted milk powder and TBS / T (137 mM NaCl and 0.05% (vol / vol /) with phosphorylation site-specific and ERK antibodies against COX-2, NFκB, ERK. It was searched in 5% (wt / vol) BSA dissolved in 20 mM Tris-HCl buffer, pH 7.6) containing vol) Tween-20. Using a horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody, bound antibody was detected by enhanced chemiluminescence. Signals were monitored using Western Lighting ™ Plus-ECL (Perkin Elmer Life Sciences) and PVDF membranes were exposed to Luminescent image analysers L'AS-3000 (Fujifilm, Minato, Tokyo). The data obtained were analyzed and the differences between treatments were compared.

The data are mean ± S. Represented as E. A statistical comparison of the results was made using one-way analysis of variance (ANOVA). The mean within each column followed by different letters was significantly different at p <0.05 by Tukey's test.
result

Western blots were used to investigate the effects of Spiranthes sinensis extract and SI on changes in COX-2, NFκB, p-ERK and ERK in LPS-induced RAW264.7 cells. Upregulation of ERK activation and COX-2 protein expression was observed at 12 hours by LPS induction in RAW264.7 cells. 12-hour LPS treatment of RAW264.7 cells increased COX-2 and NFκB expression by about 2-fold and ERK phosphorylation expression by about 250-fold. The Spirites sinensis extracts (Fig. 7) and SI (Fig. 8) show LPS-induced inflammatory cytokines and iNOs expression (LPS-induced inflammatory cytokine and iNOs expression of COX-2 and NFκB) of COX-2 and NFκB. Oxidation was reduced in a dose-dependent manner.
(Example 7)
Effect of Spiranthes sinensis extract and cinetylcarol on NO production in RAW264.7 cells Method Cell culture in LPS-induced RAW264.7 cells

The RAW264.7 mouse (murine) macrophage cell line was purchased from the Biorelocation Collection and Research Center (BCRC, Taiwan). Cells were supplemented with 100 U / mL penicillin, 100 μg / mL sodium pyruvate and 10% fetal bovine serum (FBS; Gibco Inc.) in Dalveco modified eagle medium (DMEM, Gibco Inc., NY, USA) at 37 ° C., 5 Incubated with% CO ₂ .

Cells were also treated with 0.01% DMSO as vehicle control. Cells were seeded in 12-well plates for 24 hours at a density of 6 × 10 ⁵ cells / well, then incubated in serum-free medium containing 0.1% BSA for 3 hours with various concentrations of Spiranthes sinensis extract or SI. It was treated and then incubated in the presence of LPS (1 μg / mL) for 4 hours.
Nitrite quantification

NO production from activated RAW264.7 cells was determined by measuring the amount of nitrite, NO stable oxidation product. Aliquots of conditioned medium were mixed with equal volumes of 1% sulfanilamide in water and 0.1% N-1-naphthylethylenediaminedihydrochloride in 5% phosphoric acid. The absorbance was determined at 550 nm. Sodium nitrite diluted in culture medium at a concentration of 10-100 μM was used to create the standard curve.

The data are mean ± S. Represented as E. A statistical comparison of the results was made using one-way analysis of variance (ANOVA). The mean within each column followed by different letters was significantly different at p <0.05 by Tukey's test.
result

The amount of nitrite, a stable metabolite of NO, was used as an indicator of NO production in the medium. When 1 μg / mL LPS was added, NO production increased dramatically to 2.0 ± 0.57 μM. To evaluate the effect of Spiranthes sinensis extract or SI on LPS-induced NO production in RAW264.7 cells, 12 hours Spiranthes sinensis extract (5, 20, 50 and 100 μg / mL) or SI (0.1, After treatment (1, 5 and 10 μM), cells were treated with 1 μg / mL LPS for 12 hours (FIG. 9). The Spiranthes sinensis extract and SI had an inhibitory effect on NO production in LPS-induced RAW264.7 cells in a dose-dependent manner. To ensure that plant extracts alone do not contribute to inflammation, we also tested NO production for treatment with Spiranthes sinensis extract and SI alone.
(Example 8)
In vivo antioxidant activity of Spiranthes sinensis extract Method Cell culture

The RAW264.7 mouse (murine) macrophage cell line was purchased from the Biorelocation Collection and Research Center (BCRC, Taiwan). Cells were supplemented with 100 U / mL penicillin, 100 μg / mL sodium pyruvate and 10% fetal bovine serum (FBS; Gibco Inc.) in Dalveco modified eagle medium (DMEM, Gibco Inc., NY, USA) at 37 ° C., 5 Incubated with% CO ₂ .

Cells were also treated with 0.01% DMSO as vehicle control. Cells were seeded in 12-well plates for 24 hours at a density of 6 × 10 ⁵ cells / well, then incubated in serum-free medium containing 0.1% BSA for 3 hours with various concentrations of Spiranthes sinensis extract or SI. It was treated and then incubated in the presence of LPS (1 μg / mL) for 4 hours.
DPPH assay

The DPPH radical scavenging activity of the Spiranthes sinensis extract or SI was determined as previously described (Sharma et al., 2009). Briefly, the reaction mixture in 96-well plates containing various concentrations of Spiranthes sinensis extract and 0.04 mM DPPH (SIGMA, # D9132) was incubated at 37 ° C. for 30 minutes and the absorbance was measured at 490 nm. Vitamin C was used as a positive control.
result

During inflammation, various cells, such as macrophages, undergo activation, including respiratory bursts, and a significant increase in oxygen uptake results in massive release of ROS (Yang et al., 2011). A 100 μg / mL Spiranthes sinensis extract has an antioxidant capacity comparable to 0.1 mg / mL Vitamin C (Fig. 10).
(Example 9)
THSC and NHSC cell cultures and survival NHCS and THSC cell cultures

Hepatic stellate cells (HSCs), nonparenchymal cells, are the major cell types involved in liver fibrosis and are typically involved in the formation of scar tissue in response to liver injury. In the normal liver, HSC is in quiescent form and is the main storage site for vitamin A and lipids. Vitamin A reserves gradually decrease in liver damage. When the liver is damaged, stellate cells change to an active state (Hjelkrem, Morales et al., 2012). Activated stellate cells are characterized by proliferative, contractile and chemotaxis. Therefore, the stationary form of HSC can be used as an indicator of liver condition.

As described in our previous studies, non-chemically derived hepatic stellate cells (NHSC purity> 95%) were isolated from male sprag dolly rat liver and thioacetamide (TAA) -induced hepatic stellate cells (THSC purity> 95%). %) Was isolated from the TAA (Fluka) -induced fibrous liver of male sprag dolly rats. Both cell lines were maintained in DMEM (pH 7.4) supplemented with 10% FBS and incubated in a water saturated incubator at 37 ° C. and 5% CO2. Medium was changed every 2 days and cells were passaged with trypsin / EDTA (Sigma-Aldrich) at 80% -90% confluence.
Cell viability

To examine the biocompatibility of the extracts of the compositions of the present disclosure, MTT assay (3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Invitrogen), ratio. Perform an assay based on color analysis).
MTT assay

To analyze living cells, an assay based on 3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Invitrogen), colorimetric analysis was performed. ³ cells 8 × 10 per well were seeded on 96-well plates. Cells were treated with various concentrations of Spiranthes sinensis extract or SI for 24 hours, and after incubation, 20 μL (5 mg / mL) of MTT solution per well was added and incubated for an additional 4 hours. The medium was removed and formazan was dissolved by adding 100 μL / well of DMSO (Sigma-Aldrich) and OD was measured at 570 nm using an ELISA reader. The percentage of living cells was estimated by comparing with untreated control cells. The average absorbance value of the control cells was defined as 100% survival rate.

The results in FIG. 11 showed that the extract of Spiranthes sinensis was substantially harmless to the cells at a given dose.
(Example 10)
Effect of Spiranthes sinensis extract on lipid accumulation Method

Oil Red O staining and AdipoRed assay were performed to examine the effect of the Spiranthes sinensis extract on lipid accumulation.

The data are mean ± S. Represented as E. A statistical comparison of the results was made using one-way analysis of variance (ANOVA). The mean within each column followed by different letters was significantly different at p <0.05 by Tukey's test.
Oil Red O Stain Assay

Briefly, 1 × 10 ⁵ cells per well (NHSC or THSC) were seeded in 6-well plates and incubated overnight at 5% CO2, 37 ° C. to confluence. The cells were then treated with various concentrations (5, 20 and 50 μg / mL) of this Spiranthes sinensis extract for 24 hours, respectively. Cells were washed twice with PBS and then fixed with 3.7% paraformaldehyde (preheated) for 10 minutes. A 0.5% (w / v) Oil Red O preservation solution was filtered and stained at RT in the dark for 1 hour. Cells were washed with 50% isopropanol for 5 seconds and counterstained with hematoxylin (Sigma-Aldrich) for 1 minute. Cells were washed 3 times with distilled water and stored using glycerol. Images were acquired using a ZEISS inverted microscope connected to a Canon 700 D camera.
result

The results are shown in FIG. It is noted that this Spiranthes sinensis extract increased a large amount of pernuclear lipid droplets in both NHSC and THSC when compared to the control group. The results of FIG. 12 are also quantified as the bar graph shown in FIG. 13, and from the latter, the treatment of this Spiranthes sinensis extract preserved the fatty acids of NHSC and THSC by about 3 times and about 1 respectively as compared with the untreated control group. It was further clear that the increase was 5.5 times.
AdipoRed Assay

Approximately 1 × 10 ⁴ cells per well were seeded on 96-well plates and incubated overnight at 5% CO2, 37 ° C. Cells were treated with various concentrations (5, 20, 50 and 100 μg / mL) of Spiranthes sinensis extract for 48 hours. After incubation, the culture supernatant was removed and each well was carefully rinsed with 200 μL PBS. Each well was then filled with 200 μL PBS and 5 μL AdipoRed Reagent (Lonza, Walkersville, MD, USA). After incubation at room temperature for 10 minutes, the plate was placed on a fluorometer to obtain fluorescence by excitation at 485 nm and luminescence at 572 nm (Multimode Plate Reader, PerkinElmer Inc Waltham, MA, USA).
result

The results (FIG. 14) show that the Spiranthes sinensis extract increased lipid accumulation in NHSC (1.8-fold) and THSC (1.4-fold) when compared to the control group. The observations were consistent with the results of the Oil Red O staining assay described above, further confirming the ability of this Spiranthes sinensis extract to accumulate lipids.
(Example 11)
Effect of Spiranthes sinensis on collagen accumulation Method

Sirius red staining was used to examine the effect of this Spiranthes sinensis extract on collagen accumulation. Briefly, ⁴ x 10 cells per well were seeded on 12-well plates and incubated overnight at 5% CO2, 37 ° C. to confluence. The cells were then treated with various concentrations (5, 20 and 50 μg / mL) of this Spiranthes sinensis extract for 24 hours, respectively. Cells were washed twice with PBS and then fixed with 3.7% paraformaldehyde (preheated) for 10 minutes. The cells were stained with Picro-Sirius Red solution (ScyTek, Logan, Utah, USA) at RT for 1 hour in the dark. Cells were washed twice with ddH2O and counterstained with hematoxylin (Sigma-Aldrich) for 1 minute. Cells were washed 3 times with distilled water and stored using glycerol. Images were acquired using a ZEISS inverted microscope connected to a Canon 700 D camera.

The data are mean ± S. Represented as E. A statistical comparison of the results was made using one-way analysis of variance (ANOVA). The mean within each column followed by different letters was significantly different at p <0.05 by Tukey's test.
result

The results are shown in FIG. Both NHSC and THSC treated with Spiranthes sinensis extract (5, 20 and 50 μg / mL) for 24 hours showed a reduction in perinuclear collagen compared to the control group.
(Example 12)
Effect of Spiranthes sinensis extract on the expression of COL1 I, COL1 III, TGF- / 3 R1, TGF- / 3 R2, RXRα, NrF2 and PPARγ Method

COL1 III (collagen alpha 1 type III), COL1 I (collagen alpha 1 type I), TGF-β R1 (TGF-β receptor 1), TGF-β R2 (TGF-β receptor 2), RXRa (retinoid X) Receptor), NrF2 (nuclear factor (erythrocyte-derived 2) -like 2) and PPARγ (peroxysome growth factor activating receptor y) expression was examined by real-time PCR, using 18S as a control.

For RNA isolation and cDNA synthesis, ⁵ HSCs 4x10 were seeded in 6-well plates, 70-80% confluent, and then at various concentrations in DMEM containing 5% FBS (5, 20, 50 and 100 μg / mL) of this Spiranthes sinensis extract were treated for 8 and 12 hours, respectively. The positive control group was treated with 25 μM curcumin. Total RNA was prepared from freshly recovered HSCs and isolated using trizol extraction. RNA samples were frozen at −80 ° C. until analysis. The cDNA was then prepared using the M-MLV RT kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol.

SensiFAST SYBR No-ROX Kit (BIOLINE, London, UK) was used for real-time PCR. The PCR cycling conditions for RXRα, NrF2 and PPARγ included a heating step at 95 ° C. for 2 minutes at the beginning of each run. The tube was then annealed at 95 ° C. for 5 seconds, 64 ° C. for 10 seconds, and stretched at 72 ° C. for 20 seconds for 40 cycles. The PCR conditions for collagen alpha 1 type III, collagen alpha 1 type I, TGF-β receptor 1 and TGF-β receptor 2 included a heating step at 95 ° C. for 2 minutes at the beginning of each run. The tube was then annealed at 95 ° C. for 5 seconds, annealed at 62 ° C. for 10 seconds, and stretched at 72 ° C. for 20 seconds for 45 cycles. Optical data was collected during the annealing phase in all cases. Internal standard by 18S to quantify expression for each PCR product. The primer sets used in this example are listed in Table 2.

The data are mean ± S. Represented as E. A statistical comparison of the results was made using one-way analysis of variance (ANOVA). The mean within each column followed by different letters was significantly different at p <0.05 by Tukey's test.
result

FIG. 16 shows that NHSC and THSC treated with the present Spiranthes sinensis extract for 8 hours had reduced expression of COL1 I, COL1 III, TGF-βR1 and TGF-βR2.

17 and 18 show that RXRα, NrF2 and PPARγ gene expression was increased in both NHSC and THSC treated with this Spiranthes sinensis extract for 12 hours.
(Example 13)
Effect of Spiranthes sinensis on tissue damage / wound healing Method

Rats were divided into two groups: control group animals treated with sterile gauze, and Spiranthes sinensis extract (SS) group animals treated with 5% Spiranthes sinensis extract with white petrolatum and sterile gauze. Treatment was applied to the wound to damage the skin in the dorsal region of the rat and to assess the effect of the Spiranthes sinensis extract on wound healing. The wound size (1.5 cm x 1.5 cm) of each rat was measured and recorded. Wounds are treated with control or Spiranthes sinensis extract, day 0, day 1, day 2, day 3, day 5, day 6, day 7, day 9, day 12, and day 13. Observed in. Scar tissue resection was performed on day 14 and histopathological examination was performed by staining with hematoxylin and eosin. Wound healing size and percentage were calculated using ImageJ software.
result

FIG. 19 shows the progression of wound healing in rat skin in response to treatment. Rats treated with the Spiranthes sinensis extract showed extensive gradual recovery compared to controls. Figures 20 and 21 show changes in the repair area in SS and control rats. Furthermore, this beneficial effect was demonstrated by tissue sections (Fig. 22). Rats in group SS showed completion of the skin epithelium and dermis layer in the wound area, while the skin epithelium and dermis layer were not fully healed.
(Example 13)
Preparation of Spiranthes sinensis extract

Air-dried Spiranthes sinensis was obtained from local retailers, Hualien, Taiwan. Dried Spiranthes sinensis was ground into powder and extracted with 100% ethyl acetate (preferably 10 times the dry weight) for 1 week at room temperature (RT). The liquid extract was evacuated to produce its dry powder with a yield of about 20.9% based on dry weight. Preservative compositions were made by dissolving the extract in DMSO at a concentration of 1 mg / mL based on the total volume of DMSO for subsequent experiments. Preservative compositions were made by dissolving the extract in DMSO at a concentration of 1 mg / mL based on the total volume of DMSO.
(Example 14)
Effect of Spiranthes sinensis extract on cell viability

The RAW264.7 mouse (murine) macrophage cell line was purchased from the Biorelocation Collection and Research Center (BCRC, Taiwan). Cells were supplemented with 100 U / mL penicillin, 100 μg / mL sodium pyruvate and 10% fetal bovine serum (FBS; Gibco Inc.) in Dalveco modified eagle medium (DMEM, Gibco Inc., NY, USA) at 37 ° C., 5 Incubated with% CO ₂ . Cells were also treated with 0.01% DMSO as vehicle control. Cells were seeded in 12-well plates for 24 hours at a density of 6 × 10 ⁵ cells / well and then incubated in serum-free medium containing 0.1% BSA for 3 hours at various concentrations (5) prepared in Example 13. Treatment with 10, 20, 50 and 100 μg / mL) Spiranthes sinensis extracts was then incubated in the presence of LPS (0.4 or 1 μg / mL) for an additional 4 hours.

To examine the biocompatibility of the Spiranthes sinensis extract, in this example the MTT assay (3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Invitrogen), ratio An assay based on color analysis) was performed. Briefly, ³ cells 8 × 10 per well were seeded on 96-well plates and incubated overnight at 5% CO ₂ , 37 ° C. Cells were treated with various concentrations (5, 20, 50 and 100 μg / mL) of Spiranthes sinensis extract for 24 hours, after incubation, 20 μL (5 mg / mL) of MTT solution per well was added and incubated for an additional 4 hours. .. The medium was removed, formazan was dissolved by adding 100 μL / well of DMSO (Sigma-Aldrich), and OD was measured at 570 nm using a microplate reader (ELISA reader, Thermo Labsystems). The percentage of living cells was estimated by comparing with untreated control cells. The average absorbance value of the control cells was defined as 100% survival rate. The results show that the Spiranthes sinensis extract does not harm cells at a given dose (see Figure 24).
(Example 15)
Effect of Spiranthes sinensis extract on MMP2 in cell culture

In this example, the activity of MMP2 was determined by gelatin zymography. ^Five HSCs 2 × 10 were seeded on a 6-well plate. After 70-80% confluence, cells were deficient in DMEM containing 0.1% BSA for 6 hours, then in various concentrations (5, 20, 50 and 100 μg / mL) of Spiranthes sinensis extract for 24 hours. Each was treated. Conditional medium was collected and centrifuged at 12,000 xg, 4 ° C. for 30 minutes to remove cell debris. The supernatant was collected and quantified using a Bradford dye (Bio-Rad). An 8% SDS-PAGE gel containing 10% gelatin was prepared. The protein was preheated at 55 ° C. with 2 × load dye (0.125M Tris-HCl, pH 6.8, 4% SDS, 0.04% bromophenol blue, 20% glycerol). A 7.5 μg protein sample was loaded onto the gel and electrophoretic separation was performed at 80 V for 2-3 hours. After electrophoresis, the gel was washed twice in 50 mL of 2.5% Triton X-100 per gel, then in color buffer (0.05M Tris-HCl, pH 8.8, 5 mM CaCl ₂ , 0.02%). Incubated in NaN ₃ ) at 37 ° C. for 16 hours. Finally, the gel was stained in 0.1% Coomassie blue R-250 (Bio-Rad) for 4 hours and then decolorized with fixed buffer (45% methanol, 10% acetic acid). Gels were scanned using an Epson scanner and quantified using multi-gauge software (Fujifilm).

The activity of MMP2 was quantified by a densitometer. The results are illustrated in FIG. 27 and show that MMP2 activity was reduced in both NHSC and THSC treated with 5, 20, 50 and 100 μg / mL Spiranthes sinensis extract.

Claims (34)

A composition for reducing an inflammatory response, which comprises cinetylcarol in an amount effective for reducing the inflammatory response. The composition according to claim 1, which comprises at least 4.4 μg of cinetylcarol. The composition according to claim 1, wherein the amount of cinetylcarol is between 4.4 μg and 44 μg. Reduced inflammatory response is induced nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2), interleukin-6 ( IL-6), interleukin 1β (IL-1β), interleukin 33 (IL-33), NLRP3, phosphorylated ERK (pERK), NFκB, matrix metalloproteinase-2 (MMP2), and matrix metalloproteinase-9 ( The composition according to claim 1, which is demonstrated by reduced expression or activity of one or more biological markers selected from the group consisting of MMP9). The composition of claim 1, wherein the reduction in the inflammatory response is demonstrated by a reduction in the level of one or more free radicals in the inflammatory response. The one or more free radicals are superoxide radical anion (O ₂ ⁻ .), Hydrogen peroxide (H ₂ O ₂ ), hydroxyl radical (.OH), monomorphic oxygen ( ¹ O ₂ ), peroxynitrite. The composition according to claim 5 , which is selected from the group consisting of (ONOO ⁻ ), hydrogen peroxide (NO), nitrosonium cation (NO ⁺ ), and nitroxyl anion (NO ⁻ ). The composition of claim 1, wherein the reduction in inflammatory response is demonstrated by reduction in tissue swelling. The composition according to claim 7 , wherein the tissue is selected from the group consisting of skin, epithelium, synovial tissue, tendons, cartilage, ligaments, bones, muscles, organs, dural membranes, blood vessels, bone marrow, and extracellular matrix. Stuff. A dosage form for at least one of inducing antioxidant production, reducing the inflammatory response, and promoting wound healing, which contains an effective amount of cinetylcarol and is formulated for administration to a subject. Also, the dosage form. For intravenous administration, intraarterial administration, oral administration, parenteral administration, buccal administration, topical administration, transdermal administration, rectal administration, intramuscular administration, subcutaneous administration, intraosseous administration, transmucosal administration, or intraperitoneal administration The dosage form according to claim 9 , which is formulated in. The dosage form of claim 9 , which is formulated for topical administration. The dosage form of claim 9 , which is formulated as a unit dose in liquid form, gel form, semi-liquid form, semi-solid form, or solid form. The dosage form of claim 9 , which comprises at least 4.4 μg of cinetylcarol. The dosage form of claim 9 , wherein the amount of cinetylcarol is between 4.4 μg and 44 μg. The dosage form of claim 9 , which is formulated as a unit dose. The dosage form of claim 15 , wherein the unit dose is formulated as a food product. The dosage form of claim 15 , wherein the unit dose is formulated as a beverage. The dosage form of claim 15 , wherein the unit dose is formulated as a dietary supplement. A pharmaceutical composition for reducing an inflammatory response, (a) an amount of Spiranthes sinensis extract (SSE) present in an effective amount to reduce the inflammatory response, and (b) pharmaceutically acceptable. A pharmaceutical composition comprising a carrier, wherein the SSE is obtained by extracting Spiranthes sinensis with an organic solvent and contains at least 0.1% by weight of cinetylcarol . Reduced inflammatory response is induced nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2), interleukin-6 ( IL-6), interleukin 1β (IL-1β), interleukin 33 (IL-33), NLRP3, phosphorylated ERK (pERK), NFκB, matrix metalloproteinase-2 (MMP2), and matrix metalloproteinase-9 ( The pharmaceutical composition according to claim 19 , which is demonstrated by reduced expression or activity of one or more biological markers selected from the group consisting of MMP9). The pharmaceutical composition according to claim 19 , wherein the reduction in the inflammatory response is demonstrated by a reduction in the level of one or more free radicals in the inflammatory response. The one or more free radicals are superoxide radical anion (O ₂ ⁻ .), Hydrogen peroxide (H ₂ O ₂ ), hydroxyl radical (.OH), monomorphic oxygen ( ¹ O ₂ ), peroxynitrite. The pharmaceutical composition according to claim 21 , which is selected from the group consisting of (ONOO ⁻ ), hydrogen peroxide (NO), nitrosonium cation (NO ⁺ ), and nitroxyl anion (NO ⁻ ). The pharmaceutical composition according to claim 19 , wherein the reduction in inflammatory response is demonstrated by reduction in tissue swelling. 23. The medicament according to claim 23 , wherein the tissue is selected from the group consisting of skin, epithelium, synovial tissue, tendon, cartilage, ligament, bone, muscle, organ, hard membrane, blood vessel, bone marrow, and extracellular matrix. Composition. 19. The pharmaceutical composition of claim 19 , wherein 150 g of the SSE comprises at least 250 mg of cinetylcarol. The pharmaceutical composition according to claim 19 , which is formulated for topical administration. The carrier can be intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or peritoneal. The pharmaceutical composition according to claim 19 , which is suitable for internal administration. The pharmaceutical composition according to claim 19 , which is formulated as a unit dose in liquid form, gel form, semi-liquid form, semi-solid form, or solid form. The pharmaceutical composition according to claim 19 , which is formulated as a topical cream. The use of cinetylcarol in the manufacture of dosage forms, said dosage forms used to induce antioxidant production, use. Use of cinetylcarol in the manufacture of dosage forms, said dosage forms used to reduce the inflammatory response. Use of cinetylcarol in the manufacture of dosage forms, said dosage forms used to promote wound healing. The use according to any one of claims 30 to 32 , wherein the dosage form is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The use according to any one of claims 30 to 32 , wherein the dosage form comprises a Spiranthes sinensis extract.